(Z)-N-(3-aminopropyl)-N'-9-octadecenylpropane-1,3-diamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Read-across from an OECD 422 study, involving most specific evaluation of reproductive parameters, with a structural identical substance with on average a shorter aliphatic chain length indicates a NOAEL of 30 mg/kg bw/d. Other available studies on comparable polyamines include similar OECD 422 studies and a full two-generation study and developmental toxicity studies in rat and rabbit on a structurally related dodecane dipropylene triamine branched, have also shown no indication of concern for reproductive toxicity.No adverse effects on reproductive organs were identified in the 90 day study in rats on similar product C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine. Additionally, no treatment-related changes in the estrous cycle length or sperm were apparent. A developmental toxicity study in rat, also performed with C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine, included endpoints that are relevant to assessing an effect on fertility. No effects on pre/post implantation rate, late/early resorptions, corpora lutea or number of live fetuses were seen in this study.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

18 August 2009-12 October 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

(See also Category polyamines in support of Oleyl dipropylenetriamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Coco (C12-18) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical structure, the variability of the alkyl chain length is considered to have a possible modifying activity, which is related to modification of the physiological properties of the molecule by the increase or shortening of the apolar alkyl chain part. This is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity. In series of substances that are chemically identical but differ in length of alkyl chains, those that have shorter chain length are likely to be more bioavailable compared to those with longer chain lengths. Therefore, results from the shortest chain length, in this case Coco (C12-C18 alkyl) dipropylene triamine, can be considered a worst-case approach for the longer chain lengths, specifically Oleyl (C18, unsaturated) dipropylene triamine.

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


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

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Remarks:

specific gravity 1.036

Details on exposure:

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

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

Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Details on mating procedure:

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

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

- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion the treatment phase, according to a validated method (NOTOX project 491240). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

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

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

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

The analyzed concentrations of the test samples were corrected with the mean recovery of the procedural recovery samples, because the procedural recovery samples had recoveries of 116% and 124% and the analysed concentrations of the test samples had a similar tendency. With the correction procedure it was possible to obtain adequate results for the test samples.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for at least 42 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy (Group 4 animals were not dosed on Day 9, and were sacrificed for humane reasons on Day 9).

Details on study schedule:

- Age at mating of the mated animals in the study: Approximately 14 weeks.

Remarks:

Doses / Concentrations:
0, 10, 30 and 100 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

10, and an extra 5 males for Group 1 and 4. The study included a recovery phase for males only. These animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity. The control recovery allocation animals did not undergo a recovery period, but were sacrificed together with the control main allocation animals and the Group 4 allocation animals did not undergo a recovery period, but died spontaneously or were killed in extremis on Day 9.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected based on the results of the dose range finding/MTD study with Coco dipropylene triamine (NOTOX Project 491236). See End point Study Record 7.5.1: Repeated Dose Toxicity: oral.rat_NOTOX 491236.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily (early morning/late afternoon)

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

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

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

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

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

OPHTHALMOSCOPIC EXAMINATION: No

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

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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: Group 1, 2 and 3
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength and motor activity.

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Parameters examined in all male parental animals:
testis weight, epididymis weight.
For 5 males of the control and high dose group, slides of the testes were prepared to examine staging of spermatogenesis.

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

Litter observations:

SPARAMETERS EXAMINED
Each litter was examined to determine the following, if practically possible:

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

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

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

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

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

Postmortem examinations (parental animals):

All animals were fasted overnight (with a maximum of approximately 23.5 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and animals killed in extremis were anaesthetised using iso-flurane (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated.

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

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

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

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

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

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

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

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

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

From all remaining Main males: Epididymides, Testes.

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

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

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

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

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

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

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

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

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Postmortem examinations (offspring):

SACRIFICE
Pups were killed by decapitation on Day 5 or 7 of lactation.

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

HISTOPATHOLOGY / ORGAN WEIGTHS
no.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Fisher, 1950) was applied to frequency data.

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

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

Reproductive indices:

For each group the following calculations were performed:

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

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

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

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

Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Percentage live males at First Litter Check = Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Dose descriptor:

NOEL

Remarks:

not identified

Effect level:

< 10 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

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

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

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

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

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

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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 30 other: mg/kg/day

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

Conclusions:

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

Executive summary:

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

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

Parental findings:

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

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

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

Reproductive/Developmental findings:

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

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

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

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Read-across from structural identical substance with on average a shorter aliphatic chain length (worst case in category). Consistent results from all studies within the whole group of Polyamines, indicating a no concerns for reproduction toxicity. The indicated NOAEL of 30 mg/kg is based on high maternal toxicity and mortality at the next higher dose level of 100 mg/kg bw/day.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Quality of whole database:

Justification: Physical-chemical properties of Oleyl dipropylene triamine indicate a low likelihood for exposure via inhalation. The paste has 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:

Substance is corrosive with (very) dermal uptake, and thus testing via dermal application should normally not be conducted. 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

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

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

 

For the evaluation of repeated dose toxicity of C18-unsaturated-alkyl (Oleyl) dipropylene triamine read-across is applied from C12-18-alkyl (Coco) dipropylene triamine, a structural identical substance with on average a shorter aliphatic chain length.

Within a specific chemical structure, the variability of the alkyl chain length is considered to have a possible modifying activity, which is related to modification of the physiological properties of the molecule by the increase or shortening of the apolar alkyl chain part. This is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity. In series of substances that are chemically identical but differ in length of alkyl chains, those that have shorter chain length are likely to be more bioavailable compared to those with longer chain lengths. Therefore, results from the shortest chain length can be considered a worst-case approach for the longer chain lengths. The results with Coco dipropylene triamine are thus relevant for the evaluation of Oleyl dipropylenetriamine and likely even overestimate its level of toxicity.

 

A combined repeated dose/reproduction screening toxicity study in rats according to OECD 422 has been performed with Coco dipropylene triamine. Dose levels consisted of 0, 10, 30 and 100 mg/kgbw/day. The highest dose group of 100 mg/kgbw/day was terminated on day 9 due to high toxicity (Ulceration of the stomach). No reproductive/developmental toxicity was observed at any of the other dose levels and thus a reproduction/developmental NOAEL of 30 mg/kg/day was determined.

 

No specific adverse effects on reproductive organs were identified in the 90 day study in rats (OECD 408) on structurally similar C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine. Additionally, no treatment-related changes in the oestrous cycle length or sperm were apparent in this study. A developmental toxicity study performed on Tallow dipropylene triamine also included endpoints that are relevant to assessing an effect on fertility. No effects on pre/post implantation rate, late/early resorptions, corpora lutea or number of live foetuses were seen in this study.

 

Other available studies on comparable polyamines include an OECD 422 study on Tallow tripropylene tetramine and a full two-generation study and developmental toxicity studies in rat and rabbit on a structurally related dodecane dipropylene triamine, branched (CAS 2372-82-9, not included in this dossier). These studies have also shown no indication of concern for reproductive toxicity. 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 be necessary. 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).

Effects on developmental toxicity

Description of key information

Cross-reading to a prenatal developmental toxicity study on C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine result 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 from supporting substance (structural analogue or surrogate)

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 Oleyl dipropylene triamine attached to Ch.13)
Structurally, the two linear-alkyl dipropylene triamines Oleyl dipropylene triamine and Tallow (C16-18, C18-unsaturated) dipropylenetriamine are very similar: a linear alkyl chain and a primary amine at the end, with 2 secondary amines in between separated by a propyl group. Consequently, they share the same chemical reactivity and their physico-chemical properties are very similar from which a comparable toxicological profile can be expected.
Within a specific chemical structure, the variability of the alkyl chain length is considered to have a possible modifying activity, which is related to modification of the physiological properties of the molecule by the increase or shortening of the apolar alkyl chain part. This is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and metabolism pathways, aspects that could have an impact on specific mechanisms of toxicity.
The ratio between the C16, C18 and C18:1 (C18-unsaturated) alkyl chains in the Oleyl and Tallow dipropylene triamine products show an overlap in alkyl chain of about 50%:
- Oleyl: C18:1 = 85.5%; C18 = 12.4 %; C16: 7,1%
- Tallow: C18:1 = 26.5%; C18 = 38.3 %; C16: 35.2%
The higher level of unsaturation in Oleyl based products has never shown to have an important effect. This is in agreement with the expectation that these structures do not undergo an important level of metabolism, and if metabolism occurs on the limited absorbed material, the resulting alkyl chains will fit in the physiological pool of these natural alkyl chains. Further, the relatively somewhat sorter chain lengths of C16 would make it slightly more bioavailable, in agreement to the notion that shorter alkyl chains represent a more conservative evaluation.
Consequently, the data on Tallow (C16-18, C18-unsaturated-alkyl) dipropylene triamine can be used for the read-across to Oleyl dipropylene triamine.

Reason / purpose for cross-reference:

read-across source

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

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

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 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:

Substance is corrosive with (very) dermal uptake, and thus testing via dermal application should normally not be conducted. 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

Within the category of the Polyamine studies are available that clearly demonstrate the same toxicological profile for the various substances over this category in general, and a clear lack for concern for specific developmental toxicity from the range of available relevant studies.

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

 

For the evaluation of repeated dose toxicity of C18-unsaturated-alkyl (Oleyl) dipropylene triamine read-across is applied from C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine, a structural identical substance with on average a slightly shorter aliphatic chain length. The alkyl chains (C16, C18 and C18-unsatuarted) in the Oleyl and Tallow dipropylene triamine products are very similar, and respective show an overlap in distribution of these alkyl chains of about 50%. Consequently, the results with Tallow dipropylene triamine is relevant for the evaluation of Oleyl dipropylenetriamine.          

 

A prenatal developmental toxicity study according to OECD 414 guideline and GLP compliant is available for C16-18, C18-unsaturated-alkyl (Tallow) dipropylene triamine (Tallow dipropylenetriamine). 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 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. 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.

Some additional observations that were interpreted as polydactyly were seen in all dose groups. However, these were clearly caused by mechanical damage of tissues or processing artefacts with subsequent displacement of the digits, giving the appearance of extra hind paw structures.

 

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. This data includes an OECD 422 study on Coco dipropylene triamine, and developmental toxicity studies in both rats and rabbits on C12-alky dipropylene triamine, branched. These reproduction studies have also shown no indication of concern for developmental toxicity:

 

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).

Mode of Action Analysis / Human Relevance Framework

Based on structure and mechanism of cytotoxicity, reproduction toxicity is not expected. The observed effects are local, reflecting a point-of-first-contact effect.

In physiological circumstances, the polyamines have a cationic surfactant structure (nitrogens are fully protonated)which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The apolar hydrophobic tails are pushed out of solution and easily dissolve in the lipid bilayer, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Cytotoxicity through disruption of cell membrane at exposure site will occur rather than absorption over the cell membrane. Consequently, significant placental transfer is not expected to occur.

Justification for classification or non-classification

All available data from studies involving the evaluation of reproduction and developmental parameters have not shown any indication of reproductive or developmental effects. One study indicated reproductive effects, but these were attributed to artefacts. Therefore no classification is required for this endpoint.