Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-05-14 - 2010-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

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

EC Number:

230-528-9

EC Name:

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

Cas Number:

7173-62-8

Molecular formula:

C21H44N2

IUPAC Name:

N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diamine

Details on test material:

Chemical registery number : 7173-62-8
Chemical name : (Z)-N-9-octadecenylpropane-1,3-diamine

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agreed by each registrant.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier S.A.S, Route des Chênes secs-B.P.4105-53941 LE GENEST-ST-ISLE-France
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: within ± 20% of the mean weight (no more information given)
- Housing: The animals were housed individually in IVC cages (except during mating
period where 2 females were paired with one male), type III H, polysulphone cages on Altromin saw fiber bedding
- Diet: Free access to Altromin 1324 maintenance diet for rats and mice
- Water: Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiol. controlled periodically)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 10%
- Air changes (per hr): 10 x / hour
- Photoperiod (hrs dark / hrs light): Artificial light, sequence being 12 hours light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved or suspended corn oil. The vehicle was chosen as suggested by sponsor and the test item’s solubility. The test item formulation was prepared freshly on each administration day before the administration procedure.

VEHICLE
- Lot/batch no. (if required): 058K0070 and 128K0040 (Sigma)

- Administered dose volume: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The assessment of homogeneity as well as a determination of the nominal concentration of the test item in the vehicle was performed at specified intervals. Analysis of the dose formulations of the test item in the vehicle (nominal concentration) was performed in the first and last week of the study for all doses. Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation. The determination was performed in the first and last week of the study. The dose formulation analysis was performed at BSL BIOSERVICE Scientific Laboratories GmbH under the BSL study Nr. 081567.

Details on mating procedure:

- Impregnation procedure: cohoused
- if cohoused:
- M/F ratio per cage: 1/2
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

The test item was administered to sperm positive females (presumed pregnant) from respective GD 0 to GD 19.

Frequency of treatment:

daily

Duration of test:

Duration of test: 20 days (animals were killed on day 20)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed twice daily during entire gestation period except during weekends and holidays where clinical observation was made only once. Mortality, morbidity, pertinent behavioural changes and all signs of overt toxicity were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The sperm positive females were weighed during GD 0, 3, 5, 8, 11, 14, 17 and 20. Males were not weighed in this study.

FOOD CONSUMPTION: Yes
- Food consumption of sperm positive females was measured on respective GD 3, 5, 8, 11, 14, 17 and 20.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uterus, ovaries

OTHER:
- Inflammatory Markers:
Serum samples were collected at terminal sacrifice from all females and stored at = -20 °C for the possible analysis of inflammatory markers by ELISA technique.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control and test groups. Statistical significance for fetal anamolies were determined by Chi Square analysis. Statistical analysis was performed with GraphPad Prism (Version V) software (p < 0.05 was considered as statistical significant).

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item related clinical signs were observed in HD group females during the entire treatment period. However, there were also findings like vocalisation, regurgitation, moving the bedding and salivation in few animals of MD group, which were observed transiently and occurred immediately after dose administration. These findings lasted for 1 to 3 days and later the animals recovered.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were four decedents in the study, 1 in MD group (Animal 62) and 3 in HD group (Animals 80, 82 and 98). Animal no. 82 was euthanised for humane reason. The death of two animals (Animals 62 and 98) were considered due to gavaging error and other two (Animals 80 and 82) was considered might be due to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease was observed for body weight and body weight development throughout the gestation period in HD group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Statistically significant decrease in food consumption was observed during the entire Gestation period in HD group compared to corresponding control.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Gravid uterus weight was not affected.

Maternal developmental toxicity

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

effects observed, treatment-related

Description (incidence and severity):

Decrease in pregnancy rate was observed in HD group (69.23%) as compared to LD (96%), MD (95.8%), and control (95.8%)

Details on maternal toxic effects:

Effects on maternal body weight, food consumption, clinical/ macroscopic findings and rate of pregnancy in the animals of HD group were considered to be treatment related.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

Results (fetuses)

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant deviation in sex ratio in LD and MD groups when compared to control was reported. This is not considered treatment-related.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant deviation in male litter weight and total number of male fetuses in LD group when compared to control was reported. This is not considered treatment-related.

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Few gross external anamolies were seen among the control and treatment groups. Typical external finding noted were protruding tongue, micrognathia, malrotated limbs, edematous neck, small neck, dome shaped head, swollen jaw and macrognathia. These findings had no statistical significance when compared between treatment and control groups. Hence, the findings were considered incidental.
Statistical analysis revealed significant deviation for a normal variant hematoma on right eye (LD group), right scapula (LD and MD groups), right forepaw (LD and MD groups) and right hindlimb (LD, MD and HD groups) in most of the fetuses compared to control.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Craniofacial examination by razor blade serial sectioning technique revealed no statistical significant difference for any of the findings observed in treatment and control groups.
Skeletal examination of the Alizarin red stained fetuses revealed a range of anamolies, which occurred at an incidence in both treatment and control groups. However, the statistical analysis revealed the significant differences for few variations, namely, incomplete ossification of 4th sternebrum (LD group), large nasofrontal suture (MD group), supernumerary 14th thoracic rib on right side (MD group) and bilateral sides (HD group) and small hyoid bone (HD group).

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Internal observation of the viscera by free hand micro dissection technique revealed range of visceral anamolies in all groups including control. The statistical analysis revealed the significant increase in incidences for variations such as bilaterally hemorrhagic kidney (HD group), split thymus (MD group), unilaterally (left) dilated renal pelvis (MD group), bilaterally convoluted ureter (HD group) compared to corresponding controls. There was also a statistically significant increase of minor anomaly small spleen (MD group) compared to corresponding controls.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The terminally sacrificed animals belonging to the HD group revealed incidences of few lesions at necropsy, namely, gas filled stomach and intestine, white spots on adrenals, discoloured liver, small spleen and thymus, enlarged adrenals, discoloured heart and bloody lung.

Details on embryotoxic / teratogenic effects:

There were also statistically significant increases in the incidences of few fetal effects (external, visceral and skeletal) in treatment groups compared to corresponding controls. All fetal findings except for small spleen (minor anomaly) were classified as variation.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

external malformations

skeletal malformations

visceral malformations

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Summary of clinical observations

Clinical Finding	Group
	C (0 mg/kg)	LD (1.25 mg/kg)	MD (5 mg/kg)	HD (20 mg/kg)
Total number of animals examined	24	25	25	26
Regurgitation	0	1	1	0
Dyspnoea	0	1	0	1
Moving the bedding	0	0	2	1
Salivation	0	0	3	15
Vocalization	0	0	4	9
Sneezing	0	0	0	4
Bloody nasal discharge	0	0	0	1
Weight loss	0	0	0	5
Piloerection	0	0	0	3
Half eye lid closure	0	0	0	1
Apnoea	0	0	0	2
Swollen abdomen	0	0	0	1
Swollen snout	0	0	0	1
Cyanosis	0	0	0	1
Diarrhea	0	0	0	1

Summary of prenatal data

Parameters		C (0 mg/kg)	LD (1.25 mg/kg)	MD (5 mg/kg)	HD (20 mg/kg)
Terminal Body weight (g)	Mean ± SD N	420.17 24.94 23	412.33 38.72 24	408.83 24.85 23	369.13* 53.44 16
Uterus weight (g)	Mean ± SD N	73.16 13.83 23	68.79 26.44 24	74.52 16.57 23	67.5* 24.25 16
Adjusted maternal weight (g)	Mean ± SD N	347.04 20.66 23	343.54 19.42 24	334.3 18.97 23	301.63* 34.13 16
Corpora lutea	Mean ± SD N	14.26 1.48 23	14.88 2.76 24	15.0 2.58 23	15.19 1.42 16
Implantation	Mean ± SD N	12.74 2.14 23	12.63 4.22 24	12.74 3.15 23	13.5 3.2 16
Live Fetuses	Mean ± SD N	12.22 2.26 23	11.75 4.70 24	12.74 3.15 23	11.88 4.5 16
Early resorptions	Mean ± SD N	0.48 0.79 23	0.88 1.03 24	0.52 0.9 23	0.75 1.44 16
Late resorptions	Mean ± SD N	0.04 0.21 23	0.0 0.0 24	0.0 0.0 23	0.88 3.5 16
Total resorptions	Mean ± SD N	0.52 0.79 23	0.88 1.03 24	0.52 0.9 23	1.63 3.59 16
Dead Fetuses	Mean ± SD N	0.0 0.0 23	0.04 0.2 24	0.09 0.29 23	0.0 0.0 16
Sex Ratio (M/F)	Mean ± SD N	2.27 2.37 23	0.96* 0.61 23	1.02* 0.66 22	12.67 3.31 15
Pre-implantation loss	Mean ± SD N	10.86 10.55 23	16.96 22.51 24	15.17 15.3 23	11.07 19.09 16
Post-implantation loss	Mean ± SD N	4.24 6.04 23	11.88 22.48 24	0.0 0.0 23	11.38 25.65 16

Summary of Fetal Visceral Examination

Observations	Group	Control		Low Dose		Mid Dose		High Dose
	Dose	0 mg/kg bw		1.25 mg/kg bw		5 mg/kg bw		20 mg/kg bw
	No. Of litters evaluated	23		24		23		16
	No. Of pups evaluated	147		147		152		99
		A	B	A	B	A	B	A	B
Hemorrhagic Kidney (B)		0	0.0	1*	0.75	3	2.11	5	5.49
Convoluted Ureter (B)		7	5.19	11	8.21	16	11.27	15*	16.48
Dilated Renal Pelvis (L)		0	0.0	3	2.24	5*	3.52	0	0.0
Thymus		0	0.0	2	1.49	4*	2.82	1	1.1
Small Spleen		2	1.48	2	1.49	9*	6.34	2	2.2

Summary of Fetal Skeletal Examination

Observations	Group	Control		Low Dose		Mid Dose		High Dose
	Dose	0 mg/kg bw		1.25 mg/kg bw		5 mg/kg bw		20 mg/kg bw
	No. Of litters evaluated	23		24		23		16
	No. Of pups evaluated	147		147		152		99
		A	B	A	B	A	B	A	B
Supernumerary rib-14th T (B)		6	4.08	10	6.8	24*	15.79	4	4.04
Supernumerary rib-14th T (R)		2	1.36	8	5.44	8	5.26	6*	6.06
Large-NasoFrontal Suture		0	0.0	1	0.68	5*	3.29	0	0.0
IO-4th Sternerbum		0	0.0	5*	3.4	1	0.66	0	0.0
Split-Interparietal		15	10.2	13	8.84	18	11.84	19*	19.19
Small-Hyoid		0	0.0	2	1.36	3	1.97	3*	3.03

Applicant's summary and conclusion

Conclusions:

Based on the findings, the NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg/ kg body weight based on clinical observations seen in some animals at 5 mg/kg, whereas the NOAEL for embryo-fetal toxicity is believed to be 20 mg/ kg body weight.

Executive summary:

This Prenatal developmental toxicity study of N-Oleyl-1,3-diaminopropane was conducted in pregnant female Wistar rats to detect the possible adverse effect on pregnant females and embryofetal development when administered by oral gavage from respective gestation day 0 to 19.

Nulliparous and non pregnant females were mated with males (2:1 ratio) and divided into four groups based on their body weights on day of positive vaginal smears (GD 0). Four groups of presumed pregnant females were dosed daily by oral gavage with 1.25, 5 and 20 mg/kg body weight per day of N-Oleyl-1,3-diaminopropane at dose volume of 4 mL/kg body weight. Control animals were handled identically as treated groups and received vehicle in similar volume as treated groups. The test item formulation was prepared freshly and dose volumes were adjusted based on the most recent body weight measurement. Animals were examined daily for the clinical signs and mortality. Body weight and food consumption was measured on various gestation days. The treated and control females were sacrificed on respective gestation day 20.

Followed by the gross necropsy evaluation of the females, the uteri and ovaries were removed, weighed and examined for number of implantations, resorptions (early and late) live and dead fetuses. Fetuses were identified by colour strings, sexed and weighed. All fetuses were observed for the external abnormalities, half of the fetuses for the visceral abnormalities, craniofacial examination and remain half of the litter for skeletal abnormalities. Uteri of the non pregnant females were processed with 0.5 % ammonium sulphide solution and checked for the early embryonic deaths if any.

 

Results:

Test item related clinical signs were observed in high dose females during the entire treatment period. Also effects on clinical observations were observed in animals of the MD group.

However, there were four decedents in this study [1 in MD group (Animal 62) and 3 in HD group (Animals 80, 82 and 98)]. Animal no. 82 was euthanised for humane reason. The death of two animals (Animals 62 and 98) were considered due to gavaging error and other two (Animals 80 and 82) might be considered due to toxicity. 

Statistically significant decrease was observed for body weight and body weight change throughout the gestation period in HD group.  Statistically significant decrease in overall food consumption was observed in HD group compared to corresponding control. Statistical analysis of prenatal data revealed significance in prenatal parameters like gravid uterus weight and adjusted maternal weight in HD group compared to corresponding controls. No other prenatal parameters like No. of corpora lutea, implantations percent preimplantation loss, group mean number of live fetuses, early resorptions, late resorptions, total resorptions, group mean number of female fetuses, sex ratio (M/F) and percent post implantation loss showed statistical deviation compared to corresponding controls.

 

Statistically significant difference was observed for group male litter weight (LD group), sex ratio (LD and MD groups) and total number of male fetuses (LD group) compared with controls. These findings were not attributed to toxicity as no dose related pattern was observed. Decrease in pregnancy rate was observed in HD group (69.56%) as compared to LD (96%), MD (95.8%), and control (95.8%).

Few gross external abnormalities were seen in fetuses among the control and treatment groups. Typical external findings noted were protruding tongue, malrotated limbs, micrognathia, edematous neck, small neck and hematoma (localised).But no statistical deviation was observed for these above findings except for hematoma which were localised and did not show dose related pattern.

Internal observation of the viscera by free hand micro discussion technique revealed range of visceral abnormalities in all groups including control. However, the statistical analysis revealed differences for findings viz., hemorrhagic kidney-bilateral (HD group), convoluted ureter-bilateral (HD group), dilated renal pelvis-left side (MD group), split thymus (MD), small spleen (MD group). Most of the above findings (except for hemorrhagic kidney and convoluted ureter in HD group) were not attributed to toxicity due to lack of dose dependent effect.

Craniofacial examination by razor blade serial sectioning technique revealed no statistical significant difference for any of the findings observed in treatment and control groups. 

Skeletal examination of the Alizarin red stained fetuses revealed a range of abnormalities which were of a type or which occurred at an incidence in both treatment and control groups. The statistical difference observed for supernumerary 14th rib-right side-bilaterally (MD group) and right (HD group), large naso-frontal suture (MD group), incomplete ossification of 4th sternebrum (LD group), split interparietal (HD group) and small hyoid (HD group) were attributed to toxicity, but the statistical difference observed for other anomalies were not considered of toxicological relevance due to lack of dose related pattern.

The terminally sacrificed animals belonging to the HD group revealed incidences of few lesions at necropsy, which were as gas filled stomach and intestine, whitish spots on adrenals, discoloured liver, small spleen and thymus, enlarged adrenals, bloody lung, discoloured heart, bloody lung. The finding like dark coloured food rest in caecum observed in most of the animals of control and treatment groups cannot be considered as toxicity related and in most of the animals this finding observed was not reported.

 

Based on the findings, the NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg/ kg body weight based on clinical observations seen in some animals at 5 mg/kg, whereas the NOAEL for embryo-fetal toxicity is believed to be 20 mg/ kg body weight.