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

Developmental toxicity / teratogenicity

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl -propane-1,3-diamine

EC Number:

628-863-4

Cas Number:

1219458-14-6

Molecular formula:

No molcular formula

IUPAC Name:

N-(3-aminopropyl)-N'-C16-18 (evennumbered), C18 unsaturated alkyl -propane-1,3-diamine

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Details on test material:

- Name of test material (as cited in study report): C16-18, C18-unsaturated-alkyl dipropylene triamine
- Substance type: White paste
- Physical state: Solid
- Lot/batch No.: S001096
- Expiration date of the lot/batch: 29 March 2017
- Storage condition of test material: At room temperature in the dark under nitrogen
- General information: Previously this substance was identified as Tallow dipropylenetriamine with CAS number 61791-57-9
- Reactivity: Reactive to oxygen
- Test substance handling: Flush container with nitrogen after handling
- Density: 835 kg/m3 at 60°C
- pH (1% in water, indicative range): 10.2 – 10.6 (determined at WIL Research Europe)
- Stability at higher temperatures: Yes, maximum temperature: 80°C, maximum duration: several hours
- Melting point/range: 35-45°C
- Stability in Propylene glycol: Stability for at least 5 hours at room temperature and at least 8 days at room temperature in the dark under nitrogen is confirmed over the concentration range 0.5 to 32 mg/mL

Test animals

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

Administration / exposure

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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Details on embryotoxic / teratogenic effects:
Litter size: No treatment related effect on litter size was noted up to 120 mg/kg.The mean number of viable fetuses per litter was 12.3, 12.3, 12.4 and 10.9 in the control, 30, 60 and 120 mg/kg groups, respectively.
Sex ratio: There were no treatment-related effects on the sex ratio of the fetuses.
Fetal body weight: Fetal body weights were significantly lower at 120 mg/kg compared to controls, which was secondary to the lower gains/maternal weight loss at this dose level. Body weights of fetuses (sexes combined) were 3.4, 3.4, 3.4 and 2.8 grams for the control, 30, 60 and 120 mg/kg groups, respectively.
Visceral malformations were observed in 4(3), 0(0), 4(4) and 1(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively. None of the malformations noted at 60 and 120 were considered to be treatment related.
The viscerally malformed fetus at 120 mg/kg had internal hydrocephaly which was considered to be spontaneous in origin due to its single occurrence. At 60 mg/kg, 3 fetuses from 3 litters either had an absent eye or a small eye. These eye anomalies only occurred at 60 mg/kg and therefore no dose relationship could be established. Another fetus in the mid dose group had situs inversus whereby all thoracic and abdominal organs were laterally transposed. The same finding was observed in 3 control fetuses and thus the occurrence at 60 mg/kg was not considered to be toxicologically relevant or treatment related.
Other visceral malformations in this study only affected control fetuses. Abnormal lobation of the lung was observed in two fetuses which also had situs inversus. In addition, one of these fetuses had multiple cardiovascular abnormalities (narrow pulmonary trunk, absent ductus arteriosus, malpositioned left subclavian and ventricular septum defect) and a split spleen. Another control fetus had abnormal lobation of the liver, absent lung lobe, transposition of the great vessels and ventricular septum defect.
Of the visceral variations, discolored adrenal glands were observed in 0(0), 0(0), 7(2) and 48(5) fetuses (litters) of the control, 30, 60 and 120 mg/kg groups, respectively.
Other variations noted in test substance treated groups were small supernumerary liver lobes, liver appendix, partially undescended thymus horns, convoluted ureter, dilated ureter and right subclavian originating from the aortic arch. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose-relationship and/or occurred at frequencies within the historical control range.
Skeletal malformations were observed in 7(4), 13(6), 14(9) and 3(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively.
Two malformations, polydactyly and malpositioned metatarsals, which are considered to be closely related, were observed unilaterally in the in 1(1), 6(3), 5(4) and 3(1) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively, where one fetus in the 30 mg/kg group showed both polydactyly and malpositioned metatarsals in the left hind paw.
Another malformation observed was bent limb bones. It occurred in 4(3), 8(4) and 6(3) and 0(0) fetuses (litters) in the control, 30, 60 and 120 mg/kg groups, respectively, resulting in 1.5%, 2.9%, 3.0% and 0.0% of fetuses per litter in these same respective dose groups. In all these fetuses one or both scapulas were bent and additionally, the humerus and/or radius were involved in two fetuses of Group 2 and one of Group 3.
The incidences of bent limb bones in the 30 and 60 mg/kg groups (2.9% and 3.0% per litter, respectively) were higher than the historical control data range (0.0%-1.6% per litter) and concurrent control value (1.5% per litter), but as no cases occurred in the high dose group, a relation to treatment could not be established.
Remaining skeletal malformations observed in fetuses of test substance treated groups were sternoschisis (two fetuses in one litter of Group 3) and vertebral anomaly with or without associated rib anomaly (one fetus of group 3). Because these findings occurred at a low incidence, were seen in historical controls and, in the case of vertebral anomaly with or without associated rib anomaly, occurred in a concurrent control fetus they were not considered to be treatment related.
The only other skeletal malformation in this study was noted in one control fetus. This fetus had a vertebral centra anomaly.
Skeletal variations were observed in 87.7%, 84.1%, 83.3% and 80.1% of fetuses per litter in the control, 30, 60 and 120 mg/kg groups, respectively.
Ossification parameters observed at a statistically significantly higher incidence in the 120 mg/kg group compared to the control group were unossified sternebrae nos. 5 and/or 6 (36.5% versus 13.3% per litter), unossified vertebral centra (9.2% versus 0.8% per litter) and unossified metacarpals and/or metatarsals (9.5% versus 0.0% per litter). Additionally, higher (not statistically significant) mean litter incidences were noted at 120 mg/kg compared to controls for unossified hyoid (1.5% versus 0.7% per litter), unossified sternebrae nos. 1, 2, 3 and/or 4 (8.5% versus 0.4% per litter), entire sternum unossified (3.7% versus 0.0% per litter), reduced ossification of sternebrae (18.6% versus 1.3% per litter), reduced ossification of vertebral centra (11.8% versus 1.8% per litter), reduced ossification of vertebral arches (14.2% versus 2.5% per litter), unossified or reduced ossification of pubis (7.6% versus 1.2% per litter) and unossified or reduced ossification of ischium (1.9% versus 0.4% per litter). A statistically significantly decreased incidence was observed for the finding of reduced ossification of the skull at 120 mg/kg (9.7% per litter) compared to the control value (16.5% per litter).
At 60 mg/kg, a higher incidence (not statistically significant) was noted for unossified sternebrae nos. 1, 2, 3 and/or 4 (2.3% versus 0.4% per litter), reduced ossification of sternebrae (8.4% versus 1.3% per litter), reduced ossification of vertebral arches (7.7% versus 2.5% per litter), unossified or reduced ossification of pubis (4.5% versus 1.2% per litter) and unossified or reduced ossification of ischium (2.2% versus 0.4% per litter) compared to the control group.
Decreased mean litter incidences of ossification parameters at 60 mg/kg were observed for unossified sternebrae nos. 5 and/or 6 (8.3% versus 13.3% per litter, statistically significant) and reduced ossification of the skull (8.0% versus 16.5% per litter, not statistically significant).
At 30 mg/kg, mean litter incidences of ossification parameters listed in text table 2 were comparable to the control values.
Of the other skeletal variations, bent ribs were noted in 21.8%, 23.7%, 25.8% and 4.6 % of fetuses per litter in the control, 30, 60 and 120 mg/kg groups, respectively. The value at 120 mg/kg was statistically significantly decreased compared to the control value, but was within the historical control data range (1.6%-27.4% per litter). A reduced number of fetuses with bent ribs is not an adverse effect and has no toxicological significance. Therefore, the decreased mean litter incidence of bent ribs was considered to have arisen by chance and was not considered to be treatment related.
A statistically significantly decreased mean litter incidence of 14th rudimentary ribs was noted at 120 mg/kg compared to the control value (31.3% versus 61.0% per litter). At 30 and 60 mg/kg, 47.3% and 48.7% of fetuses per litter, respectively, had 14th rudimentary ribs. All values were within the historical control data range (17.9%-72.4% per litter) and because there was no dose-dependent relationship noted for other supernumerary ribs (14th full ribs, 7th cervical full ribs and 7th cervical rudimentary ribs (see table 1 and 2), the decrease of 14th rudimentary ribs at 120 mg/kg was not considered to be treatment related.
Remaining skeletal variations noted in this study were ossified cervical centrum no. 1, slightly to moderately malaligned sternebrae, supernumerary ossification site in sternum, branched sternebrae, caudal shift of pelvic girdle and reduced ossification of ribs. These variations were not considered to be treatment related, because they occurred at similar frequencies in the control group, occurred infrequently, occurred without dose-relationship and/or occurred at frequencies within the historical control range.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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

Executive summary:

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

 

Maternal findings

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

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

 

Developmental findings

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

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

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

 

External malformations and variations:

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

 

Visceral malformations and variations:

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

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

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

 

Skeletal Malformations and Variations:

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

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

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

 

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

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

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

 

 

Discussion reported skeletal malformations

Malformations	fetuses
Dose level	control	30 mg/kg	60 mg/kg	120 mg/kg
Number examined skeletally	270	259	236	217
Polydactyly
no. of fetuses (litters)	0(0)	5(3)	1(1)	2(1)
% per litter	0	1.7	0.4	0.7
fetuses affected	-	A023-09,12 A032-11,12 A044-09	A057-12	A080-01,09
Metatarsal(s)- Malpositioned
no. of fetuses (litters)	1(1)	2(1)	4(3)	1(1)
% per litter	0.4	0.7	1,5	0,3
fetuses affected	A001-10	A023-10,12	A046-09 A048-09,12 A053-03	A080-11

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

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

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

 Polydactyly (external examination): 1 fetus

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

 

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

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

 

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

 

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

 

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

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

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