Amines, N-(3-aminopropyl)-N'-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

No reproductive/developmental toxicity was observed at any dose level with read-across substance Coco dipropylenetriamine. This product is considered to be linked with higher toxicity related increased bioavailability based on somewhat shorter average alkyl chain lengths while actual chemical structure and related biochemical activity is the same. A reproduction/developmental NOAEL of 30 mg/kg/day was determined, based on termination of the 100 mg/kg dose group between study days 6-9 due to severe toxicity and mortality.

No adverse effects on reproductive organs were identified in the 90-day study in rats on C16-18, C18-unsaturated-alkyl dipropylene triamine itself. Additionally, no treatment-related changes in the estrous cycle length or sperm were apparent. Developmental toxicity studies performed with the substance in rats and rabbits 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 fetuses were seen in these studies. In addition, there is no consumer exposure to C16-18, C18-unsaturated-alkyl dipropylene triamine, and manufacture and use are highly controlled, limiting the possibility of exposures.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels (migrated information)

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

18 August 2009-12 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. Read-across from structural identical substance with on average a shorter aliphatic chain length.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
See analogue approach justification report attached to chapter 13.2 Other Assessment Reports.

1. HYPOTHESIS FOR THE CATEGORY APPROACH (ENDPOINT LEVEL)
For the evaluation of repeated dose toxicity ofC16-18, C18-unsaturated-alkyl dipropylene triamine, also called Tallow dipropylenetriamine, read-across is applied from Coco dipropylenetriamine, a structural identical substance with on average a shorter aliphatic chain length thus representing a conservative approach.

2. ANALOGUE APPROACH JUSTIFICATION (ENDPOINT LEVEL
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 metabolisation 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 dipropylenetriamine are thus relevant for the evaluation of Tallow dipropylenetriamine and likely even overestimate its level of toxicity.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

reference to same study

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:

Consistent results from all studies within the whole group of Polyamines, indicating no concerns for reproduction toxicity. The indicated NOAEL of 30 mg/kg is based on high maternal toxicity and mortaility at the next higher dose level of 100 mg/kg bw/day.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Effect on fertility via oral route:

For the evaluation of repeated dose toxicity ofC16-18, C18-unsaturated-alkyl dipropylene triamine, also called Tallow dipropylenetriamine, read-across is applied from Coco dipropylenetriamine, a structural identical substance with on average a shorter aliphatic chain length thus representing a conservative approach.

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 dipropylenetriamine are thus relevant for the evaluation of Tallow 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 dipropylenetriamine. 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 Tallow dipropylenetriamine. Additionally, no treatment-related changes in the estrous cycle length or sperm were apparent in this study. Developmental toxicity studies in rats and rabbits performed with Tallow dipropylenetriamine 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 these studies.

 

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 branched dipropylene triamine. 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 further reproduction studies are 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).

 

Effect on fertility via inhalation route: 

Physical-chemical properties of C16-18, C18-unsaturated-alkyl 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:

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.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study in rats resulted to a maternal NOAEL for C16-18, C18-unsaturated-alkyl dipropylene triamine 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. A subsequent prenatal developmental toxicity study in rabbits resulted to a maternal NOAEL of 10 mg/kg/day, based on mortality and effects on body weights and food consumption at 20 mg/kg/day. The developmental NOAEL was established as being 10 mg/kg/day. The developmental data from the 20 mg/kg/day group was not used for any conclusions as with 15 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached. However, the available results from this dose level do not indicate developmental effects also at 20 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21-Apr-2021 to 08-Dec-2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

Official Journal of the European Union No. L142, May 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Chatillon sur Chalaronne, France)
- Arrival: Untreated females will be mated at the Supplier and will be at Day 0 or 2 post-coitum on
arrival at the Test Facility (Day 0 post-coitum is the day of successful mating). The actual age
of animals received will be listed in the Final Report.
- Age at study initiation: 17 – 20 weeks old
- Weight at study initiation: Approximately 3000 to 4300 g
- Fasting period before study: No fasting, See Diet
- Housing: Cages with perforated floors (Ebeco, Germany, dimensions
67 x 62 x 55 cm) equipped with water bottles.Animals individually housed. Cages will be arranged on the racks according to a Latin-square model.
- Diet (e.g. ad libitum): KLIBA NAFAG Rabbit Diet 3409 maintenance and breeding, from
Granovit AG, Kaiseraugst, Switzerland. Pellets. Restricted access. On arrival animals will receive approximately 25 grams pelleted diet and on subsequent days 140-160 grams will be supplied.
- Water (e.g. ad libitum): Municipal tap water. Freely available to each animal via water bottles. Periodic analysis of the water is performed, and results of these analyses are on file at the Test Facility. It is considered that there are no known contaminants in the water that could interfere with the outcome of the study.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): The actual daily mean temperature during the study period was 18 to 19°C
- Humidity (%): actual daily mean relative humidity of 48 to 61%
- Air changes (per hr): At least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12-hours light and 12-hours dark (may be interrupted for designated procedures)

IN-LIFE DATES: From: To: 19April2021 - 21May2021

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Frequency: Once daily
Duration: Day 7 to Day 28 post-coitum.
Formulations were heated to a maximum temperature of maximally 60°C for a maximum of 25 minutes to obtain visual homogeneity. Formulations were released for dosing when they have obtained a temperature of 40°C or lower.
Formulations (w/w) were prepared at least weekly, homogenized to visually acceptable levels and stored at room temperature (15-25°C) under nitrogen until dosing. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing.
Adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test item.
Method: The dose volume for each animal were based on the most recent body weight measurement. The doses were given using a plastic catheter attached to a plastic disposable syringe. Dose pot identification via Provantis was used as additional check to verify the dosing procedure according to Standard Operating Procedures.

VEHICLE
- Concentration in vehicle: 0, 4, 8 and 16 mg/mL for vehicle, 5, 10 and 20 mg/kg bw/day groups.
- Amount of vehicle: 1.25 mL/kg bw
- Justification for use and choice of vehicle (if other than water): Based on trial formulations conducted by study facility and approved by study sponsor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

During week 1 of treatment dose formulation samples were collected for analysis.

For concentration analysis, samples from the middle part of the dosing container of all dose groups were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For concentration, all mean sample concentration results within or equal to ± 10% (solutions) ± 15% (suspensions) of theoretical concentration were considered acceptable.

For homogeneity analysis, samples from the top, middle and bottom parts of the dosing container of groups 2 and 4 (low- and high-dose groups) were taken (2 x approximately 500 mg). Samples were stored at a temperature set to maintain 18-22°C. For homogeneity, a relative standard deviation (RSD) of concentrations of ≤10% for each group was considered acceptable.

Stability analyses performed previously in conjunction with the method development and validation study (ABL Study No. 501610) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study.

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 17-20 weeks
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage. 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.
- Any other deviations from standard protocol: no

Duration of treatment / exposure:

Day 7 to day 28 post-coitum.

Frequency of treatment:

once daily 7d/wk

Duration of test:

from Day 0 to Day 29 (scheduled euthanasia)

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Control

Dose / conc.:

5 mg/kg bw/day (actual dose received)

Remarks:

Low-dose group

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Remarks:

Mid-dose group

Dose / conc.:

20 mg/kg bw/day (actual dose received)

Remarks:

High-dose group

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 20279148)
- 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: mortality was checked at least twice daily beginning upon arrival through termination/release. Except on days of receipt and necropsy where frequency were at least
once 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; starting on Day 7 post-coitum up to the day prior to necropsy.
Observations will be made postdose. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

BODY WEIGHT
- Time schedule for examinations: On Days 7, 9, 12, 15, 18, 21, 24, 27 and 29 post-coitum.

FOOD CONSUMPTION
- Daily from Day 3 post-coitum onwards.

WATER CONSUMPTION
Regular basis throughout the study. Water consumption will be monitored by visual inspection of the water bottles. If inter group differences are noted, consumption may be assessed by weight.

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:

Each ovary and uterine horn of all animals was dissected and examined as quickly as
possible to determine the following as part of the necropsy procedure:
• The number of corpora lutea.
• The weight of the uterus (not for animals found dead, sacrificed before planned
necropsy or that started to deliver).
• The number of implantation sites.
• The number and distribution of live and dead fetuses.
• The number and distribution of early and late resorptions

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:

All statistical tests were be conducted at the 5% significance level. All pairwise comparisons
were conducted using two sided tests and are reported at the 1% and 5% levels, unless
otherwise noted.

- Parametric/Non-Parametric: Levene’s test will be used to assess the homogeneity of group variances. The groups will be compared using an overall one-way ANOVA F-test if Levene’s test is not significant or the Kruskal-Wallis test if it is significant. If the overall F-test or Kruskal-Wallis
test is found to be significant, then pairwise comparisons will be conducted using Dunnett’s
or Dunn’s test, respectively.

- Non-Parametric: The groups will be compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test is found to be significant, then the above pairwise comparisons will be conducted using Dunn’s test.

- ANCOVA: The data corresponding to a response variable of interest and to a related covariate will be submitted to an analysis of covariance (ANCOVA), including only groups with at least three
non-missing paired values and if found to be significant, then pairwise comparisons will be conducted using Dunnett’s test.

- Incidence: A Fisher’s exact test will be used to conduct pairwise group comparisons of interest.

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):

In total, 19/22 females treated at 20 mg/kg/day survived until scheduled necropsy of which
14/19 females were dosed for the entire treatment period.
Due to overall clinical condition of five females treated 20 mg/kg/day, it was decided to allow
a dosing holiday (for 2 to 4 days) for these females. This temporary dosing intermission was
initiated for Female Nos. 67, 76, 79 and 84 from 12 May 2021 onwards (Days 21 to 24
post-coitum, depending on mating date) until recovery in body weight and/or food
consumption was observed again. Female No. 78 was not dosed on Day 24 post-coitum, due
to its clinical signs.
Considering that this pause in dosing occurred after completion of the organogenesis,
generally considered the period between Days 6-18 post-coitum, and based on clinical signs
the animals have been dosed up to their respective maximum tolerability also after this
period, it is considered that the results from the evaluation of the litters of these animals
remain valid for the evaluation for developmental toxicity of the substance up to maximum
tolerable dose levels.
Clinical signs observed for females treated at 20 mg/kg/day were considered test item-related
unless stated otherwise.
- Pregnant females surviving until scheduled necropsy -
At 20 mg/kg/day, a thin appearance was observed for Female No. 68 on Days 26 to 27.
Reduced feces production was observed in 11/20, 8/20, 16/20 and 9/10 pregnant females in
the control, 5, 10 and 20 mg/kg/day groups, respectively. As this occurred in all groups,
including control, at comparable incidences and based on the time of onset and duration this
was considered unrelated to treatment with the test item.
Any other clinical signs noted during the treatment period occurred within the range of
background findings to be expected for rabbits of this age and strain which are housed and
treated under the conditions in this study and did not show any apparent dose-related trend. At
the incidence observed, these were considered to be unrelated to treatment with the test item.
These clinical signs included fur loss, small skin lesions on mouth or muzzle, scabs on lip,
muzzle or nose, swollen muzzle, and broken teeth.
- Non-Pregnant females surviving until scheduled necropsy -
Erected fur was observed for Female No. 72 on Day 26 post-coitum.
Reduced feces production was observed 2/2, 1/1 and 3/4 non-pregnant females in the control,
10 and 20 mg/kg/day groups, respectively. As this occurred in groups, including control, at
comparable incidences and based on the time of onset and duration this was considered
unrelated to treatment with the test item.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
Erected fur was observed for 3/4 pregnant females (up to seven consecutive days). One
pregnant female was additionally observed with a thin appearance (Days 25 to 27 postcoitum) and pale feces (Days 25 to 28 post-coitum), whereas another pregnant female was
observed with hunched posture (Days 24 and 25 post-coitum).
Female No. 78 was apathic on Day 24 post-coitum and was therefore not dosed as fixation for
dosing was unsuccessful (recorded in study daybook).
Female No. 79 (non-pregnant) was observed with abnormal breathing sounds and erected fur.
Other clinical signs included reduced and/or absent feces production for all females with a
dosing holiday and scabs, which were considered unrelated to treatment with the test item.
Females treated at 20 mg/kg/day sacrificed in extremis
Erected fur was observed in 2/3 females (up to five consecutive days). Female No. 83 was
observed with wheezing and labored breathing on Day 13 post-coitum.
Other clinical signs included reduced feces production for 2/3 females and an injured right
eye, including a superficial ulcer on the cornea and conjunctivitis for one female. These
clinical signs were considered unrelated to treatment with the test item.

Mortality:

mortality observed, treatment-related

Description (incidence):

Six females were sacrificed during the study period, of which three (high dose) were
considered test item-related, and three (low or mid dose) were considered unrelated to
treatment with the test item.
At the moment of early termination, all females, except Female No. 83, were gravid.
Test item-related mortality at 20 mg/kg/day:
Female No. 83 (20 mg/kg/day) was sacrificed in extremis on Day 13 post-coitum, based on
clinical signs.
Female Nos. 74 and 82 were sacrificed in extremis on Days 22 and 19 post-coitum,
respectively, due to effects on food consumption and body weight.
As findings were in line with the those of surviving animals, clinical signs, body weight, food
consumption and macroscopic findings of these moribund animals are mentioned in the
sections below).
Mortality unrelated to treatment with the test item:
Female No. 42 (5 mg/kg/day) was sacrificed on Day 8 post-coitum because of poor
acclimatization after arrival. Food consumption was minimal to absent (ranging between
0-6 grams feed per day) during the acclimatization period (Days 3-6 post-coitum) and after
initiation of treatment (Day 7 post-coitum), and body weight loss (2% compared to Day 7
post-coitum; 10% compared to Day 0 post-coitum, non-GLP) was observed. Clinical signs
included decreased fecal production on Days 7 and 8 post-coitum. No macroscopic findings
were observed.
Female No. 30 (5 mg/kg/day) was sacrificed in extremis on Day 9 post-coitum as a result of
the oral gavage procedure. Directly after dosing, the animal showed labored breathing and the
animal was necropsied after consulting the veterinarian. At necropsy, brown-gritty fluid was
found in the thoracic cavity, red fluid in the trachea and the lungs were dark-red discolored.
Body weight and food consumption were considered normal.
Female No. 64 (10 mg/kg/day) was sacrificed on Day 27 post-coitum for animal welfare
reasons, due to a necrotic lesion with discharge (40 x 30 mm) in the cervical ventral region
observed from Day 25 post-coitum onwards. Other clinical signs included scabs and
decreased fecal production. Food consumption was reduced from Day 22 post-coitum
onwards but was considered normal in the periods before. Normal body weight gain was
observed from Day 7 to 24 post-coitum, with a slight body weight loss (2%) on Day 27
post-coitum.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Pregnant females surviving until scheduled necropsy -
At 20 mg/kg/day, mean body weight gain over the total study period (Days 7-29 post-coitum)
was within the same range as the control group. However, a mean lower body weight gain
(not statically significant) was observed compared to control between Days 7-9 post-coitum.
At 5 and 10 mg/kg/day, mean body weight gain was comparable to concurrent control.
The higher body weight gain observed over Day 7-9 post-coitum in females treated at
5 mg/kg/day was considered to be unrelated to treatment with the test item as no trend was
apparent regarding dose and duration of treatment.
Mean body weights of the control group were higher (not statistically significant) compared
to the 5, 10 and 20 mg/kg/day groups (up to 6%) upon randomization and at the initiation of
dosing, which resulted in mean body weights that were statistically significantly lower on
Days 12, 27 and 29 post-coitum at 10 mg/kg/day and from Day 9 post-coitum onwards at
20 mg/kg/day.
Body weight gain adjusted for the gravid uterus was considered unaffected up to
20 mg/kg/day.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
For 2/5 females, body weight loss up to 9% compared to their maximum body weight was
observed, resulting in a dosing holiday for several days. After this dosing holiday body
weight gain recovered to normal.
For 2/5 females, a stable body weight was observed during the treatment period, with one of
these females losing 4% of its weight between Days 27 and 29 post-coitum.
For 1/5 females, normal body weight gain was observed.
- Females treated at 20 mg/kg/day sacrificed in extremis -
2/3 females were observed with body weight loss up to 7 to 9% compared to the initiation of
treatment. 1/3 females had a stable body weight prior to sacrifice.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Pregnant females surviving until scheduled necropsy -
Mean food consumption was considered unaffected upon treatment up to 20 mg/kg/day.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
Overall, food consumption was lower compared to normal values (between 140-160 gram per
day) for all 5 females, with various periods showing a severely reduced food consumption
(≤20 grams per day) for 4/5 females. In 2/5 females, food consumption recovered to normal
values after the dosing holiday was initiated.
- Females treated at 20 mg/kg/day sacrificed in extremis -
Food consumption was reduced in 3/3 females during of the treatment period. Two females
were observed with a reduced food consumption from the initiation of treatment, developing
in an absent food consumption (0-13 gram per day) from Day 12 or 14 post-coitum onwards.
For the other female, food consumption was considered to be within normal ranges during the
first days of treatment, but a reduced food consumption prior to necropsy was observed.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- Pregnant and non-pregnant females surviving until scheduled necropsy -
At 10 mg/kg/day, one pregnant female was observed with mucoid white content on the
fundus wall of the stomach and dark-black foci on the gallbladder wall. As the mucoid white
content on the stomach wall was also observed in one non-pregnant female treated at
20 mg/kg/day (as well as dark-black foci on the stomach), a test item-relation could not be
excluded.
Macroscopic observations at necropsy did not reveal any alterations that were considered to
have arisen as a result of treatment with the test item in the remaining animals.
Findings among test item-treated animals included dark-black foci on the lungs, prominent
lobular architecture of the liver and/or gray and hard nodule in the liver. In the absence of a
dose response and/or occurrence of the finding in the control group as well, they were
considered changes of no toxicological significance.
- Females treated at 20 mg/kg/day with a dosing holiday up to four days -
No macroscopic abnormalities at necropsy were observed.
- Females treated at 20 mg/kg/day sacrificed in extremis -
For 1/3 females, dark-black foci on the glandular stomach and mucoid white content on the
fundus wall were observed, which was considered test item-related.
The eye-injury of 1/3 females was confirmed at necropsy, which was considered unrelated to
the test item. For 1/3 females, no macroscopic findings were observed.

Details on results:

In total, 19/22 females treated at 20 mg/kg bw/day survived until scheduled necropsy of which 14/19 females were dosed for the entire treatment period.
Due to overall clinical condition of five females treated 20 mg/kg bw/day, it was decided to allow a dosing holiday (for 2 to 4 days) for these females. This temporary dosing intermission was initiated for 4 females until recovery in body weight and/or food consumption was observed again. One female was not dosed on Day 24 post-coitum, due to its clinical signs.
Considering that this pause in dosing occurred after completion of the organogenesis, generally considered the period between Days 6-18 post-coitum, and based on clinical signs the animals have been dosed up to their respective maximum tolerability also after this period, it is considered that the results from the evaluation of the litters of these animals remain valid for the evaluation for developmental toxicity of the substance up to maximum tolerable dose levels.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The mean numbers of corpora lutea and implantation sites in the control, 5 and 10 mg/kg bw/day groups were comparable and in the range of normal biological variation.

At 20 mg/kg bw/day pre- and post-implantation loss was comparable to that observed for the control group and in the range of normal biological variation.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The mean numbers of pre- and post-implantation loss in the control, 5 and 10 mg/kg bw/day groups were comparable and in the range of normal biological variation.

At 20 mg/kg bw/day two females scored as non-pregnant were observed with corpora lutea (one which had a dosing holiday and one which was sacrificed in extremis). In rabbits it is hard to distinguish between non-pregnancy and early resorption of the litter that occurred during the first days after implantation, as they are both presenting as an empty uterus. As the total number of females with litters was reduced at the dose level on which severe maternal toxicity was observed, it cannot be excluded that possible implantation points were resorbed in a very early stage of the pregnancy and therefore were not visible during necropsy.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females, including those that were necropsied preterm, were found to be pregnant, except for two females of the control group, two of the 5 mg/kg bw/day, one female of the 10 mg/kg bw/day and five females of the 20 mg/kg bw/day. Of these females, three were observed with corpora lutea (one control female: 2 corpora lutea, and two females of the 20 mg/kg bw/day having 3 or 13 corpora lutea).
Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5, 10 and 20 mg/kg bw/day, respectively. Of these high dose group females, four had a temporary dosing intermission of up to four days during the study between Days 21 and 28 post-coitum, depending on mating date.
At 20 mg/kg bw/day, the number of pregnant females at necropsy was remarkably lower compared to concurrent control and historical control data (Historical Control Data, Albino, New Zealand White Rabbit (2016-2020): No. of Animals Examined at Laparohysterectomy = 729; No. Non-gravid = 61).

Details on maternal toxic effects:

All females, including those that were necropsied preterm (see Section 7.2.1), were found to
be pregnant, except for Female Nos. 09 and 16 (control group), Nos 27 and 41 (5 mg/kg/day),
No. 48 (10 mg/kg/day) and Nos. 72, 73, 79, 83 and 86 (20 mg/kg/day). Of these females,
three were observed with corpora lutea (control Female No. 9; 2 corpora lutea, No. 79;
3 corpora lutea and No. 83; 13 corpora lutea, both treated at 20 mg/kg/day).
Excluding non-pregnant females and females that did not survive until scheduled necropsy,
there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5,
10 and 20 mg/kg/day, respectively. Of these high dose group females, Nos. 67, 76, 78 and 84
had a temporary dosing intermission of up to four days during the study between Days 21 and
28 post-coitum, depending on mating date.
The developmental data from the 20 mg/kg/day group was not used for any conclusions and
was reported separately to avoid misinterpretation as surviving females did not meet the minimum number of 16 surviving to termination with implantations. Below an overview is given of
the females of which the data could not (fully) be used for a rough, overall description of the
developmental data at 20 mg/kg/day. Breakdown of Females in the 20 mg/kg/day group (Group 4) of which Developmental Data were not used for any Conclusions.
Female No. Reason for Interpretation of the Data with Caution/Exclusion:
F67 Temporarily dosing intermission Not dosed on Days 24, 25, 27 and 28 post-coitum; F72 Not pregnant – Data excluded; F73 Not pregnant – Data excluded; F74 Euthanized in extremis on Day 22 post-coitum – Data excluded; F76 Temporarily dosing intermission Not dosed on Days 23 and 26 post-coitum; F78 Temporarily dosing intermission Not dosed on Day 24 post-coitum; F79
Temporarily dosing intermission – Data excluded Not dosed on Days 22, 23 and 25 post-coitum
Not pregnant; F82 Euthanized in extremis on Day 19 post-coitum – Data excluded; F83 Euthanized in extremis on Day 13 post-coitum – Data excluded Not pregnant; F84 Temporarily dosing intermission Not dosed on Days 21, 22, 24 and 25 post-coitum; F86 Not pregnant – Data excluded.
The mean numbers of corpora lutea and implantation sites, and pre- and post-implantation
loss in the control, 5 and 10 mg/kg/day groups were comparable and in the range of normal
biological variation.
At 20 mg/kg/day, the number of pregnant females at necropsy was remarkably lower
compared to concurrent control and historical control.
Two females scored as non-pregnant were observed with corpora lutea (No. 79 which had a dosing holiday and No. 83 which was sacrificed in extremis). In rabbits it is hard to distinguish between non-pregnancy and early resorption of the litter that occurred during the first days after
implantation, as they are both presenting as an empty uterus. As the total number of females
with litters was reduced at the dose level on which severe maternal toxicity was observed, it
cannot be excluded that possible implantation points were resorbed in a very early stage of
the pregnancy and therefore were not visible during necropsy.

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Remarks on result:

other:

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

There were no test item-related relevant effects on fetal body weights (both sexes) noted by
treatment up to 10 mg/kg/day. Note: At 20 mg/kg/day, the mean litter size was considered within normal values.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 10 mg/kg/day.
Note: At 20 mg/kg/day, the male:female ratio was considered normal

Changes in litter size and weights:

not specified

Description (incidence and severity):

There were no test item-related relevant effects on fetal body weights (both sexes) noted by
treatment up to 10 mg/kg/day.
At 20 mg/kg/day, mean fetal body weights (both sexes) were lower compared to control,
reaching statistical significance for male weights only.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No external variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
One control fetus, No. 22-L9, was observed with an inwards malrotated right hind-paw and
one fetus at 5 mg/kg/day (No. 34-R8) which was dead, had a distended abdomen. In addition,
one late resorption of the control group (No. 15-R7) had gastroschisis. Based on the single
occurrence and/or the occurrence in the control group only, these were considered to be
unrelated to treatment with the test item. The dead fetus at 5 mg/kg/day (No. 34-R8) that had a distended abdomen was also observed with the external variation: “localized subcutis, edema”. Its single incidence rules out a test item-effect.
At 20 mg/kg/day, no external variations or malformations were observed

Skeletal malformations:

not specified

Description (incidence and severity):

No skeletal variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
Skeletal malformations were discovered in the ribs and vertebra in 3 (3), 10 (8) and
2(2) fetuses (litters) of the control, 5 and 10 mg/kg/day groups, respectively. These were
observed at low incidences and/or not in a dose dependent manner; consequently, all were
ruled out as test-item related.
All skeletal variations observed up to 10 mg/kg/day, occurred in the absence of a dose-related
incidence trend and/or infrequently or occurred in conjunction with a malformation.
Therefore, they were considered not test item-related.
At 20 mg/kg/day, skeletal malformations were discovered in the ribs and vertebra in 4 (3)
fetuses (litters).
Noteworthy were the skeletal variations “unossified hind-paw phalanges” and “misaligned
sternabra”, that had a statistical significant increased incidence compared to concurrent
control.
For the variation “un-ossification of hind-paw phalanges”, this increase was caused by three
fetuses out of three litters (Nos. 67-L1, 77-R10 and 88-L3). Two of these fetuses, which were
observed with low body weights as well (17.7 and 21.7 grams), showed additional signs of
retarded ossification for the talus and sternebrae. These findings are in line with the slightly
lower group mean fetal weights at this dose level.
The increase for “misaligned sternebra” was not accompanied by an increased misalignment
of other bones i.e. ilium, costal cartilage and vertebra.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No visceral variations or malformations that were considered to be test item-related were
observed up to at least 10 mg/kg/day.
Visceral malformations were observed in 3 (2), 2 (2) and 2 (2) fetuses (litters) of the control,
5 and 10 mg/kg/day groups, respectively. As the malformations occurred in single fetuses
and/or in control fetuses only, these were considered spontaneous events and not test
item-related.
Visceral variations occurred across a variety of structures, occurring at low incidences and/or
in the control group only, ruling out any test item-relationships. The statistically significant
increase in the incidence of “retrocaval right ureters” observed at 5 mg/kg/day was considered
not related to treatment with the test item in the absence of a dose dependency.
At 20 mg/kg/day, visceral malformations were observed in 2(1) fetuses (litters).

Details on embryotoxic / teratogenic effects:

Note: As according to the guidelines, a minimum of 16 females with implantations at
termination is required in each group for an adequate evaluation, the 15 such animals
available at 20 mg/kg/day were considered not sufficient for a robust and valid evaluation of
the developmental data. As such, the developmental data from the 20 mg/kg/day group cannot
be used for any conclusions. However, despite formally one litter short, it is important to note
that even at 20 mg/kg bw/day, which is possibly just above the maximally tolerated level, still
the only developmental effects observed involved a slight increase of skeletal variations.
These involved “unossified hind-paw phalanges” and “misaligned sternabra” and are in
general in line with the also observed slightly lower group mean fetal weights at this dose
level.No test item-related changes were noted up to 10 mg/kg/day in any of the developmental
parameters investigated in this study (i.e. litter size, sex ratio, fetal body weights, external,
visceral and skeletal malformations and developmental variations).

Key result

Dose descriptor:

NOAEL

Effect level:

> 10 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other:

Remarks on result:

other: Possible reduction observed at 20 mg/kg bw/day. Though based off guidance dose group does not qualify for conclusions.

Key result

Abnormalities:

no effects observed

Developmental effects observed:

not specified

DOSE FORMULATION ANALYSES

Table 1 - Accuracy and Homogeneity Test

Group	Sample Position	Concentration^1 [mg/mL]		Accuracy [%]		Homogeneity [%]
		Target	Analyzed	Individual	Mean
1	50% height	0 0	nd nd	na na	na	na
2	90% height   50%height   10% height	4 4 4 4 4 4	4.24 4.19 4.39 4.25 4.25 4.23	106 105 110 106 106 106	106	1.6
3	50% height	8 8	9.19 8.64	115 108	111	na
4	90% height   50%height   10% height	16 16 16 16 16 16	17.7 17.3 17.9 17.5 16.6 16.9	111 108 112 109 104 106	108	2.8

¹ Due to issues with the analytical system, sample extracts prepared on 26 Apr 2021 were re-diluted and analyzed on 30 Apr 2021. The sample extracts were stored in normal laboratory conditions at room temperature from 26 Apr 2021 until analysis on 30 Apr 2021.

n.d. Not detected.

n.a. Not applicable.

Accuracy
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The determined mean values ranged from 106% to 111%. No test item was detected in the Group 1 (vehicle) formulation.

Homogeneity
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%). The determined mean values ranged from 1.6% to 2.8%.

 

 

Table 2 - Summary of Body Weights: Gestation (mean values, grams)

	Day(s) Relative to Mating
	0	7	9	12	15	18	21	24	27	29
0 mg/kg bw/day	3764.0	3717.4	3743.0	3807.1	3893.9	3947.7	3978.2	4025.1	4074.3	4116.1
5 mg/kg bw/day	3615.4	3561.4	3639.9	3665.2	3757.4	3808.1	3856.7	3915.0	3974.6	4033.8
10 mg/kg bw/day	3490.6	3491.5	3524.9	3574.8*	3675.9	3728.3	3778.6	3810.6	3819.7*	3864.4*
20 mg/kg bw/day	3498.8	3486.1	3485.7*	3513.1**	3586.6**	3593.8**	3621.0**	3722.1*	3771.9**	3822.0*

Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01

 

Table 3 - Summary of Food Consumption: Gestation (mean values, g/animal/day)

	Day(s) Relative to Mating
	7 to 9 [G]	9 to 12 [G1]	12 to 15 [G1]	15 to 18 [G]	18 to 21 [G]	21 to 24 [G1]	24 to 27 [G1]	27 to 29 [G]	7 to 29 [G1]
0 mg/kg bw/day	133.88	129.38	102.62	116.30	130.73	116.10	98.32	104.28	116.21
5 mg/kg bw/day	143.29	138.15	109.39	129.33	131.72	120.85	103.98	113.14	123.31
10 mg/kg bw/day	140.69	129.86	111.32	129.46	137.46	111.78	72.21*	90.28	114.85
20 mg/kg bw/day	122.26	104.84*	83.45	93.08	97.25	102.64	89.82	95.80	102.41

[G] - Kruskal-Wallis & Dunn
[G1] - Anova & Dunnett: * = p ≤ 0.05

 

Table 4 - Summary of Maternal Performance and Mortality

		0 mg/kg bw/day	5 mg/kg bw/day	10 mg/kg bw/day	20 mg/kg bw/day
Group size – females		22	22	22	22
Number of Females Pregnant	N %	20 90.9	20 90.9	21 95.5	17 77.3
Female with Live Fetuses	N %	20 100.0	20 100.0	21 100.0	17 100.0
Total Resorptions	N %	0 0.0	0 0.0	0 0.0	0 0.0
Female with all Nonviable	N %	0 0.0	0 0.0	0 0.0	0 0.0
Terminal Euthanasia	N %	22 100.0	20 90.9	21 95.5	19 86.4
Unscheduled Death/Euthanasia	N %	0 0.0	2 9.1	1 4.5	3 13.6
Unscheduled Euthanasia	N %	0 0.0	2 9.1	1 4.5	3 13.6

 

Table 5 - Summary of Ovarian and Uterine Examinations and Litter Observations

		0 mg/kg bw/day	5 mg/kg bw/day	10 mg/kg bw/day	20 mg/kg bw/day
Female with Live Fetuses	N %	20 100.0	18 100.0	20 100.0	15 100.0
Number of Corpora Lutea [k]	Mean %Diff	11.4 -	11.3 -0.1	10.9 -4.4	11.1 -1.9
Number of Implantations [k]	Mean %Diff	10.5 -	10.6 1.1	9.6 -8.6	10.6 1.0
Pre-implantation Loss (%) [k]	Mean %Diff	7.21 -	6.33 -12.13	11.98 66.16	4.86 -32.61
Total Number of Fetuses [k]	Mean %Diff	9.9 -	9.9 0.4	8.9 -10.6	10.1 1.7
Number of Live Fetuses [k]	Mean %Diff	9.9 -	9.9 -0.1	8.9 -10.6	10.1 1.7
Number of Dead Fetuses [k]	Mean %Diff	0.0 -	0.1 -	0.0 -	0.0 -
Number of Early Resorptions [k]	Mean %Diff	0.5 -	0.4 -11.1	0.6 20.0	0.2 -60.0
Number of Late Resorptions [k]	Mean %Diff	0.1 -	0.2 122.2	0.2 50.0	0.3 233.3
Total Number of Resoprtions [k]	Mean %Diff	0.6 -	0.7 11.1	0.8 25.0	0.5 -11.1
Post-implantation Loss (%) [k]	Mean %Diff	5.40 -	6.77 25.23	7.98 47.70	4.45 -17.62
Number of Live Male Fetuses [k]	Mean %Diff	4.5 -	5.3 18.5	4.6 1.1	5.0 11.1
Number of Live Feale Fetuses [k]	Mean %Diff	5.4 -	4.6 -15.6	4.3 -20.4	5.1 -6.2
Live Male Fetus/Litter (%) [k]	Mean %Diff	46.80 -	53.23 13.74	50.17 7.20	49.92 6.67
Live Female Fetuses/Litter (%)	Mean %Diff	53.20 -	46.15 -13.25	49.83 -6.33	50.08 -5.86
Mean Fetal Weight Males (g) [G]	Mean %Diff	40.71 -	41.07 0.89	41.19 1.18	35.94* -11.72
Mean Fetal Weight Females (g) [G]	Mean %Diff	38.83 -	39.16 0.86	39.75 2.36	36.12 -6.98
Mean Fetal Weight all (g) [G]	Mean %Diff	39.61 -	40.36 1.89	40.46 2.15	35.95 -9.24

[k] - Kruskal-Wallis & Dunn

[G] - Anova & Dunnett: * = p ≤ 0.05

Conclusions:

The prenatal developmental toxicity study with C16-18, C18-unsaturated-alkyl dipropylene triamine in time-mated female New Zealand White rabbits resulted to a maternal NOAEL of 10 mg/kg/day, based on mortality and effects on body weights and food consumption at 20 mg/kg/day. The developmental NOAEL was 10 mg/kg/day being the highest dose available for evaluation.
The developmental data from the 20 mg/kg/day group was not used for any conclusions as with 15 evaluable litters the minimum required number of 16 litters for a robust and valid evaluation of the developmental data was not reached. However, the available limited results from this dose level do not indicate developmental effects.

Executive summary:

This study was conducted according to GLP in line with TG 414. Time-mated female New Zealand White rabbits were treated with C 16-18, C18-unsaturated-alkyl dipropylene triamine from Day 7 to 28 post-coitum, inclusive by daily oral gavage at dose levels of 5, 10 and 20 mg/kg/day. The rabbits of the control group received the vehicle, propylene glycol, alone.
Test formulations prepared were considered homogeneous at the concentrations tested and analysis of the accuracy revealed acceptable levels.

In total, 19/22 females treated at 20 mg/kg/day survived until scheduled necropsy. Of these, five females required a dosing pause for one to up to four days between Days 21 and 28 post-coitum due to overall very poor clinical condition. Considering that this pause in dosing occurred after completion of the organogenesis, generally considered the period between Days 6-18 post-coitum, and based on clinical signs the animals have been dosed up to their respective maximum tolerability also after this period, it is considered that the results from the evaluation of the litters of these animals remain valid for the evaluation for developmental toxicity of the substance up to maximum tolerable dose levels.

At 20 mg/kg/day, three females were sacrificed in extremis based on body weight loss, reduced food consumption and/or clinical signs. For one of these females, the observed macroscopic findings (dark-black foci on the glandular stomach and mucoid white content on the fundus wall) were considered test item-related. Comparable clinical signs and effects on body weight and/or food consumption were observed for approximately 25% of the remaining females, and therefore resulting in a dosing holiday for these females to prevent early termination. As these findings were in line with the females that were early terminated, these effects were considered to be adverse.

At 5 and 10 mg/kg/day, no test item-related clinical signs or effects on body weight and food consumption were observed. Macroscopic findings observed in a single female treated at 10 mg/kg/day were considered non-adverse due to its single occurrence and in the absence of other clinical symptoms for this animal.

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. corpora lutea, implantation sites and pre- and post-implantation loss). This thus resulted in the conclusion of a maternal NOAEL of 10 mg/kg bw/day.

Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5, 10 and 20 mg/kg/day, respectively. As according to the guidelines, a minimum of 16 females with implantations at termination is required in each group for an adequate evaluation, the 15 such animals available at 20 mg/kg/day were considered not sufficient for a robust and valid evaluation of the developmental data. As such, the developmental data from the 20 mg/kg/day group cannot be used for any conclusions. However, despite formally one litter short, it is important to note that even at 20 mg/kg bw/day, which is possibly just above the maximally tolerated level, still the only developmental effects observed involved a slight increase of skeletal variations (79.85%, 81.10%, 82.66% and 84.80% in control, 5, 10 and 20 mg/kg respectively). These involved “unossified hind-paw phalanges” and “misaligned sternabra” and are in general in line with the also observed lower group mean fetal weights at this dose level. At 20 mg/kg/day, mean fetal body weights (both sexes) were lower compared to control, reaching statistical significance for male weights only. No other notable effects were observed in dose groups 5 and 10 mg/kg/day that were attributed to dosing. This thus results in an effective developmental NOAEL of 10mg/kg/day. 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

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.

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: One animal (no. 56) died spontaneously on Day 18 and another (no. 64) was killed in extremis on Day12 of the post coitum period.

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 beginning Days 12 and Day 17 of the post coitum period, respectively, and persisted through the remaining duration of the treatment period. On day 20, the body weights were 25% lower for females at 120 mg/kg and 7% for females at 60 mg/kg compared to control.
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.

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.

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.

MATERNAL DEVELOPMENT TOXIICTY:
There were 22, 21, 21, and 21 pregnant females in the control, 30, 60 and 120 mg/kg groups, respectively, with 22, 21, 19 and 18 litters available for evaluation. There were two females at 120 mg/kg with implantation sites only (nos. 83 and 87).

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 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 female nos. 83 and 87 who both had 100% post-implantation loss, consisting of early resorptions.

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal 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.

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.

EXTERNAL MALFORMATIONS AND VARIATIONS
No external malformations and developmental variations were observed in any of the fetuses, thus it was considered that test substance treatment at dose levels up to 120 mg/kg had no effect on fetal external morphology.

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.
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 AND VARIATIONS
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.
Microscopic evaluations lead to the observation that were at first described as skeletal malformations polydactyly and malpositioned metatarsals, which are considered to be closely related, 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. However, no limb malformations were reported at external examination. Upon examination the deformation rather looked like disconnection of all toes with subsequent slight lateral displacement. on initial inspection, it would appear that short and/or supernumerary digits were present, which could reasonably be reported as ”polydactyly".
As appearance differs from polydactyly as normally described, shows an inverted dose-response relation (highest incidence at lowest dose), has no plausible mode of action resulting to a standalone effect, and is sometimes seen during instruction training for these studies as results of improper processing of foetuses, it was concluded that these findings were not true malformations but were artefacts, attributable to tissue mechanical damage or processing artefacts and subsequent displacement of the digits, which could give the appearance of extra hind paw structures.

Malformations further observed involved 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:

male/female

Basis for effect level:

fetal/pup body weight changes

Remarks on result:

other: 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, and retarded skeletal ossification seen at 60 and 120 mg/kg

Abnormalities:

not specified

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The maternal NOAEL for C16-18, C18-unsaturated-alkyl dipropylene triamine was established as being 30 mg/kg based on mortality and decreased BW at 60 and 120 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. 

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 considered 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" (disconnected toes)
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

 

The limb malformations, described as "polydactyly", "Matatarsal(s)-malpositioned" or "bent l;imb bone(s)" were reported only at skeletal examination. No limb malformations were reported at external examinatin. Upon examination the deformation rather looked like disconnection of all toes with subsequent slight lateral displacement. on initial inspection, it would appear that short and/or supernumerary digits were present, which could reasonably be reported as ”polydactyly".

Actual polydactyly is very rare. In 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 finding but artefact following processing of the fetuses during removal of the skin. 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; highest incidence at  lowest dose group. (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 genotoxicity or aneugenicity, not observed in vitro for this substance, can be expected to lead to more effects (syndromes) rather that a very specific single sided occurrence of polydactyly. ‘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:

The study is GLP compliant and has Klimisch score 2 in view of disputable interpretation of skeletal observations by the contract lab. No findings indicating developmental toxicity were seen in the OECD 414 study on the same substance in NZW rabbits (see Additional Information).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Effect on developmental toxicity: via oral route:

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

 

In the first OECD 414 study, 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 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.

 

However, there is disagreement in the interpretation between the performing contract lab and an external, independent consultant on some observations seen at skeletal evaluations. These were considered by the lab as malformations and described as "polydactyly", ”metatarsal(s) malpositioned” or ”bent limb bone(s)” were reported only at skeletal examination. No limb malformations were reported at external examination. Upon examination the deformation rather looked like disconnection of all toes with subsequent slight lateral displacement. on initial inspection, it would appear that short and/or supernumerary digits were present, which could reasonably be reported as ”polydactyly". At the request of the Sponsor, the independent external consultant examined the affected foetuses. 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.

As appearance differs from polydactyly as normally described, shows an inverted dose-response relation (highest incidence at lowest dose), has no plausible mode of action resulting to a standalone effect, and is sometimes seen during instruction training for these studies as results of improper processing of foetuses, it is considered as an artefact.

 

However, to address this divergence in interpretations, ECHA requested a second OECD 414 study to be  conducted on a second species. New Zealand White rabbits were assigned to four dose groups, each containing twenty-two animals. The test item was administered once daily by gavage from Day 7 to 28 post-coitum at doses of 5, 10 and 20 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.

In total, 19/22 females treated at 20 mg/kg/day survived until scheduled necropsy. Of these, five females required a dosing pause for one up to four days between Days 21 and 28 post-coitum due to very poor clinical condition. Considering that this pause in dosing occurred after completion of the organogenesis, generally considered the period between Days 6-18 post-coitum, and based on clinical signs the animals have been dosed up to their respective maximum tolerability also after this period, it is considered that the results from the evaluation of the litters of these animals remain valid for the evaluation for developmental toxicity of the substance up to maximum tolerable dose levels.

At 20 mg/kg/day, three females were sacrificed in extremis based on body weight loss, reduced food consumption and/or clinical signs. For one of these females, the observed macroscopic findings (dark-black foci on the glandular stomach and mucoid white content on the fundus wall) were considered test item-related. Comparable clinical signs and effects on body weight and/or food consumption were observed for approximately 25% of the remaining females, and therefore resulting in a dosing holiday for these females to prevent early termination. As these findings were in line with the females that were early terminated, these effects were considered to be adverse.

At 5 and 10 mg/kg/day, no test item-related clinical signs or effects on body weight and food consumption were observed. Macroscopic findings observed in a single female treated at 10 mg/kg/day were considered non-adverse due to its single occurrence and in the absence of other clinical symptoms for this animal.

No test item-related changes were noted in any of the remaining maternal parameters investigated in this study (i.e. corpora lutea, implantation sites and pre- and post-implantation loss). This thus resulted in the conclusion of a maternal NOAEL of 10 mg/kg bw/day.

Excluding non-pregnant females and females that did not survive until scheduled necropsy, there were 20 females with viable litters in the control group, and 18, 20 and 15 females at 5, 10 and 20 mg/kg/day, respectively. As according to the guidelines, a minimum of 16 females with implantations at termination is required in each group for an adequate evaluation, the 15 such animals available at 20 mg/kg/day were considered not sufficient for a robust and valid evaluation of the developmental data. As such, the developmental data from the 20 mg/kg/day group cannot be used for any conclusions. However, despite formally one litter short, it is important to note that even at 20 mg/kg bw/day, which is possibly just above the maximally tolerated level, still the only developmental effects observed involved a slight increase of skeletal variations (79.85%, 81.10%, 82.66% and 84.80% in control, 5, 10 and 20 mg/kg respectively) in line with the also observed lower group mean fetal weights at this dose level. At 20 mg/kg/day, mean fetal body weights (both sexes) were lower compared to control.

No other notable effects were observed in dose groups 5 and 10 mg/kg/day that were attributed to dosing. This thus results in an effective developmental NOAEL of 10 mg/kg/day. 

 

 

Studies on structurally similar substances show no indication of concerns regarding developmental toxicity. No developmental toxicity was observed in an OECD 422 screening study with Coco dipropylenetriamine. Other available studies on comparable polyamines include a similar OECD 422 study on Tallow tripropylenetetramine, and full developmental toxicity studies in rat and rabbit on a structurally related dodecane dipropylene branched triamine 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.

 

Effect on developmental toxicity: via inhalation route: 

Physical-chemical properties of of C16-18, C18-unsaturated-alkyl 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 developmental toxicity: via dermal route:

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.

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.

The mode of action of for follows from its structure, consisting of an apolar fatty acid chain and a polar part of the amines.In physiological circumstances the nitrogens can become positively charged, resulting to a cationic surfactant structure which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. At pH below 4.8 more than 99% of the substance has all three nitrogens charged positive. The apolar tails easily dissolve in the membranes, 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 uptake followed by 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. Therefore no classification is required for this endpoint.