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

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A reliable Two-Generation Toxicity to Reproduction study was conducted on the analogue substance, N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine (CAS 2372-82-9). Under the conditions of the study, 9 mg/kg bw/day (actual dose received) produced only minor parental toxicity but no reproductive effects. At 27 mg/kg bw/day (actual dose received), there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested.

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 November 1993 to 01 August 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: (P) 4 weeks; (F1) 25 d
- Weight at study initiation: (P) ca. 90 g
- Housing: Initially 2 per cage in popypropylene cages with stainless steel grid bottoms, mesh tops and food hoppers (42 x 27 x 20 cm). 3 d prior to mating the males were transferred to individual cages. For mating the females were transferred to the cage of the appropriate co-group male. Mated females were transferred to individual solid bottomed cages of the same dimensions, where they remained with the litters until termination. F1 animals selected as parents for the next generation were housed 2 per grid bottomed cage, sexes separated, and then followed the same caging regime as P animals.
- Diet: Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, ad libitum
- Water: Domestic mains water, ad libitum
- Acclimation period: 13 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared at intervals of up to 1 week. For the high dose concentration, the test substance was weighed into a labelled dose container and the requisite quantity was added and mixed by inversion as required. For the lower dose level, formulation was by serial dilution of the next highest concentration. A quitable aliquot of each formulation was dispensed to the animal room for dosing on that day. Solutions were used within 8 days of preparation.

VEHICLE
- Concentration in vehicle: 0, 1, 3 and 8 mg/mL (for 30.2% aqueous solution of the test substance)
- Amount of vehicle (if gavage): 10 mL/kg

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: Until mating or 7 nights elapsed
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear, referred to as Day 0 of pregnancy
- After 7 d of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: No
- After successful mating each pregnant female was caged (how): Individual solid bottomed cages (42 x 27 x 20 cm)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On 6 occasions during the study, triplicate samples of dosing solutions were taken and analysed for concentration and homogeneity. The method of analysis was reported the provision of IRI Project 370340.

Duration of treatment / exposure:

Parental: 10 weeks prior to mating to weaning of F1 generation
F1: From Day 25 after birth to weaning of F2 generation.

Frequency of treatment:

Daily

Details on study schedule:

F1 parental animals not mated until 11 weeks after weaning

Dose / conc.:

10 mg/kg bw/day (nominal)

Remarks:

30.2% aqueous solution

Dose / conc.:

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

Dose / conc.:

30 mg/kg bw/day (nominal)

Remarks:

30.2% aqueous solution

Dose / conc.:

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

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

30.2% aqueous solution

Dose / conc.:

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

No. of animals per sex per dose:

28/sex for parental generation and 24/sex for F1 generation

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on evaluation of existing data.
- Rationale for animal assignment (if not random): Computer generated randomly sequenced number, but ensuring that siblings were not placed in the same treatment group.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily, plus at the beginning of each day and as late as possible for viability.

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to commencement of treatment, then on each day of dosing.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded weekly for each animal, commencing 1 week prior to treatment. Food consumption monitoring was suspended during the mating period and then, for males, recommenced as before. For females, consumption was measured over Days 0-7, 7-14 and 14-20 of gestation, and Days 0-7 and 7-14 of lactation.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: No

PARAMETERS EXAMINED
The following parameters were examined in F1/F2 offspring: Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS: Yes where appropriate, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals, termination after weaning of the litters
- Maternal animals: All surviving animals, termination after weaning of the litters

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs were weighed: Ovaries, uterus, cervix and vagina, testes (weighed individually), epididymides (weighed individually), seminal vesicle, coagulating gland, prostate gland, pituitary gland. The female reproductive tract was examined for signs of pregnancy and the number of visible implantation sites was recorded.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 7 weeks of age without necropsy.

GROSS NECROPSY
Offspring found dead or killed before Day 14 of lactation were sexed, examined for the externally visible abnormalities and for the presence of milk in the stomach. Offspring dying on or after Day 14 were subjected to a gross necropsy, in which the cranial, thoracic and abdominal contents were examined macroscopically. From each litter of F1 and F2 pups, 2 male and 2 female pups were necropsied at weaning. The necropsy consisted of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. The remaining pups (except F1 weanlings selected for rearing to produce the next generation) were killed after external examination and the carcasses discarded without necropsy.

Statistics:

Where considered appropriate to assist with interpretation, statistical analysis was applied to determine the statistical significance of differences from control. Organ weight data were analysed by analysis of variance. Testes and epididymides weights were also analysed by analysis of covariance using the terminal body weight as a single covariate. For prostate and seminal vesicles weights, tests for linearity of relationship to body weight and for homogeneity of slopes indicated that analysis of covariance was inappropriate for these organs. Treatment means were compared using an F-protected Least Significant Difference procedure (Snedecor and Cochran, 1980). For other parameters, interpretation was based on examination of the individual and group values.

Reproductive indices:

The following reproductive indices were calculated for each group: Fertility index and gestation index

Offspring viability indices:

The following offspring viability indices were calculated for each litter and group: Birth index, live birth index, viability index, lactation index and overall survival index.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 27 mg/kg bw/day, most animals of both generations showed signs of reaction to treatment, consisting of dyspnea, piloerection and hunched posture, and many animals also had episodes of post-dosing salivation. For occasional animals, the outline of the spine was prominent. A total of 8 animals died or were killed after showing marked signs of reaction, and a 9th death may also have been related to treatment.
At 9 mg/kg bw/day occasional animals in both generations showed post dosing salivation; this observation was probably associated with treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In both generations, at 27 mg/kg/day, mean weight gain was markedly lower than control; this effect was apparent for males, and for females in the premating period and during gestation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption in both generations was slightly lower than in controls at 27 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating performance, fertility, duration of gestation, litter size and pup survival were considered to be similar in all groups.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on observed signs of toxicity and a marked reduction in food consumption and body weight gain of males and of females during the premating and gestation periods at 27 mg/kg bw/day.

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No adverse effects on fertility up to the highest tested dose.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Two pups at 27 mg/kg bw/day showed body tremors in late lactation; these tremors may have been associated with treatment.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 27 mg/kg bw/day, mean litter and pup weights of the F2 pups were slightly lower than controls. Although these differences were probably incidental, the possibility that they were related to treatment could not be entirely discounted. The litter and pup weights of the F1 pups at 27 mg/kg bw/day and of both generations at 3 and 9 mg/kg bw/day were similar to controls.

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity / systemic toxicity

Generation:

F2

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Based on slightly reduced pup and litter weights and observed clinical signs (2 pups with body tremors) at 27 mg/kg bw/day.

Reproductive effects observed:

no

Conclusions:

The study was performed in accordance with the guideline specified.
The performance of the 2 generations was very similar.
Under the conditions of the study, 9 mg/kg bw/day (actual dose received) produced only minor parental toxicity but no reproductive effects.
At 27 mg/kg bw/day (actual dose received), there was very marked parental toxicity, a possible minor effect on pup and litter weights, and 2 pups with body tremors. There were no obvious adverse effects on mating and littering performance at any of the levels tested.

Executive summary:

An oral two-generation study was conducted to assess the reproductive toxicity potential of N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine in rats according to the OECD Guideline 416 and in compliance with GLP.

 

Sprague Dawley rats were dosed orally by gavage, once daily at dose levels, 0, 10, 30 and 90 mg/kg/day (i.e., equivalent to 0, 3, 9 and 27 mg a.i./kg bw/day). F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were dosed for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The F1 animals were exposed to possible effect of the test substance from conception through to weaning. Clinical observations were performed daily. Body weight and food consumption were recorded at various intervals throughout the study. Females were allowed to litter normally and observations on the females and litters were recorded. All F0 and F1 animals were subjected to necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Gross lesions were monitored and applicable organs were weighed. Only 2 male and 2 female pups that were weaned from each litter underwent necropsy. No histopathology was performed, as this was available from a 90-day repeat oral dose study.

 

Toxicity to parent (adult) animals at 27 mg a.i./kg bw/day was indicated in both generations by a marked reduction in body weight gain of males, and of females during the pre-mating and gestation periods. Food consumption at this level was slightly lower than control. Additionally most animals in both generations receiving 27 mg a.i./kg bw/day showed clinical signs of reaction to treatment (principally dyspnoea, piloerection and hunched posture, many animals also had episodes of post dose salivation, and for occasional animals the outline of the spine was prominent). A total of 8 animals (2 F0 males, 3 F0 females, 1 F1 male and 2 F1 females) died or were killed after showing marked signs of reaction, and a ninth death (of an F1 female) may also have been related to treatment. At 9 mg a.i./kg bw/day, the only finding that was considered to have been probably associated with treatment was occasional animals in both generations with post dosing salivation; this finding might not be indicative of systemic toxicity.

 

Mean seminal vesicle weights in both generations at 27 mg a.i./kg bw/day were significantly lower than control; it was considered that this reduction was an indirect effect of the lower body weights rather than a direct effect on the seminal vesicles. Mating performance, fertility, duration of gestation, litter size and pup survival were considered similar in all groups of both generations. At 27 mg a.i./kg bw/day, mean litter weight and pup weights of the F2 pups were slightly lower than control; although these differences were probably incidental, the possibility that they indicated a slight effect of treatment could not be entirely discounted. The litter and pup weights of the F1 pups at 27 mg a.i./kg bw/day, and of both generations at 3 and 9 mg a.i./kg bw/day, were similar to control. Two pups at 27 mg a.i./kg bw/day showed body tremors in late lactation; these may have been associated with treatment. At the 3 mg a.i./kg bw/day dose level no marked toxicity was noted.

 

Hence, under the conditions of the study, the NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

27 mg/kg bw/day

Study duration:

subchronic

Experimental exposure time per week (hours/week):

0

Species:

rat

Quality of whole database:

The study is GLP compliant and has Klimisch score 1. The study was based on N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine and the derived NOAEL for toxicity to fertility is calculated to the Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Oral route

No adverse effects on reproductive organs were identified in the various studies available on Oleyl-diamine and its different salts and C12-14-diamine, including the 90-day study on C12-14-diamine.

 

An oral two-generation study was conducted to assess the reproductive toxicity potential the analogue substance CAS 2372-82-9 in rats according to the OECD Guideline 416 and in compliance with GLP.

Sprague Dawley rats were dosed orally by gavage, once daily at dose levels, 0, 10, 30 and 90 mg/kg/day (i.e., equivalent to 0, 3, 9 and 27 mg a.i./kg bw/day). F0 animals were randomised into the 4 treatment groups, each containing 28 males and 28 females. From each treatment group, 24 male and 24 female F1 weanlings were selected for rearing to maturity and mating to produce the F2 generation. The F0 animals were dosed for 10 weeks prior to mating, and then throughout the mating, gestation and lactation periods until sacrifice at the time of weaning of the F1 animals. The F1 animals were exposed to possible effect of the test substance from conception through to weaning. Clinical observations were performed daily. Body weight and food consumption were recorded at various intervals throughout the study. Females were allowed to litter normally and observations on the females and litters were recorded. All F0 and F1 animals were subjected to necropsy, consisting of an external examination, followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities. Gross lesions were monitored and applicable organs were weighed. Only 2 male and 2 female pups that were weaned from each litter underwent necropsy. No histopathology was performed, as this was available from a 90-day repeat oral dose study.

Toxicity to parent (adult) animals at 27 mg a.i./kg bw/day was indicated in both generations by a marked reduction in body weight gain of males, and of females during the pre-mating and gestation periods. Food consumption at this level was slightly lower than control. Additionally most animals in both generations receiving 27 mg a.i./kg bw/day showed clinical signs of reaction to treatment (principally dyspnoea, piloerection and hunched posture, many animals also had episodes of post dose salivation, and for occasional animals the outline of the spine was prominent). A total of 8 animals (2 F0 males, 3 F0 females, 1 F1 male and 2 F1 females) died or were killed after showing marked signs of reaction, and a ninth death (of an F1 female) may also have been related to treatment. At 9 mg a.i./kg bw/day, the only finding that was considered to have been probably associated with treatment was occasional animals in both generations with post dosing salivation; this finding might not be indicative of systemic toxicity.

Mean seminal vesicle weights in both generations at 27 mg a.i./kg bw/day were significantly lower than control; it was considered that this reduction was an indirect effect of the lower body weights rather than a direct effect on the seminal vesicles. Mating performance, fertility, duration of gestation, litter size and pup survival were considered similar in all groups of both generations. At 27 mg a.i./kg bw/day, mean litter weight and pup weights of the F2 pups were slightly lower than control; although these differences were probably incidental, the possibility that they indicated a slight effect of treatment could not be entirely discounted. The litter and pup weights of the F1 pups at 27 mg a.i./kg bw/day, and of both generations at 3 and 9 mg a.i./kg bw/day, were similar to control. Two pups at 27 mg a.i./kg bw/day showed body tremors in late lactation; these may have been associated with treatment. At the 3 mg a.i./kg bw/day dose level no marked toxicity was noted.

Hence, under the conditions of the study, the NOAEL for reproductive toxicity was established at 27 mg a.i./kg bw/day and the NOAEL for parental and developmental toxicity was established at the next tested lower dose level of 9 mg a.i./kg bw/day.

 

Inhalation route

Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is solid at room temperature or formulated as a liquid with pasty elements. The vapour pressure is less than 0.0015 Pa at 20°C (value is an overestimation as it is based on read-across from shorter chain C12-14-diamine). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur.

Dermal route

Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is severely irritant to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying toxicity to reproduction.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414, GLP) based on read-across from Oleyl-diamine (values converted on mw basis from Oleyl-diamine to Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates salt): Developmental NOAEL: 27 mg/kg bw, Maternal toxicity NOAEL = 1.7 mg/kg bw.

Link to relevant study records

Reference

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

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

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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)

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

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).

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.

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.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

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.

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

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

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

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

27 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is GLP compliant and has Klimisch score 1. The study was based on Oleyl-diamine and the derived NOAEL for embryo-fetal toxicity is calculated to the Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates.

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

 

Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is a salt of N-(C16-18 and C18-unsatd. alkyl)trimethylenediamine and acetic acid, in a molar ratio of 1:2. Absorption and subsequent possible toxicity follows the dissociation of the salt. The diamine structure is much more toxic than the acid part, and therefore it is considered that toxicity profile of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates will be fully driven by its tallow-diamine content. This is supported by data from repeated dose studies performed on diamine diacetates and on diamine. For further evaluation of toxicity of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, including reproduction toxicity, cross-reading can be done to the data available on diamines. For developmental DNEL, setting use is made of a developmental toxicity/teratogenicity study performed on oleyl diamine according to theOECD 414 guideline and in compliance to GLP. Pregnant rats were dosed from day 0-19 of gestation with 0, 1.25, 5 or 20 mg/kg bw of Oleyl-diamine. The animals were then sacrificed and the fetuses subject to external, visceral and skeletal examination. There was a slight increase in visceral and skeletal variations. No malformations were observed. The variations were not considered to be of toxicological relevance as there was no dose-response pattern. Maternal toxicity including deaths in the high dose group, reduced bodyweight gain and reduced pregnancy rate were observed. The NOAEL (No observed adverse effect level) for maternal toxicity is believed to be 1.25 mg Oleyl-diamine/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.

When converted to the corresponding values for the diacetate salt of talow-diamine, the levels become (Mw oleyl-diamine = 316, Mw tallow-diamine diacetate= 430):

- NOAEL maternal toxicity = 1.7 mg/kg bw/day (1.25 mg on Oleyl-diamine level)

- NOAEL embryo-fetal toxicity = 27 mg/kg bw/day (20 mg on Oleyl-diamine level)

Justification for selection of Effect on developmental toxicity: via inhalation route:
Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is solid at room temperature or formulated as a liquid with pasty elements. The vapour pressure is less than 0.0015 Pa at 20°C (value is an overestimation as it is based on read-across from shorter chain C12-14-diamine). Also the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalation route will be unlikely to occur.

Justification for selection of Effect on developmental toxicity: via dermal route:
Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates is severely irritant to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying toxicity to reproduction.

Justification for classification or non-classification

Adequate study data is only available for developmental toxicity from a valid OECD 414 guideline compliant study, showing no specific concerns, and for toxicity to fertility from a valid OECD 416 study, showing no specific concerns. Further information from repeated dose studies up to 90-days OECD 408 performed on C12/14-diamine showed no indication of concern with respect to reproductive organs evaluated.

In addition, there is no consumer exposure to Amines, N-(C16-18 and C18-unsatd.alkyl)trimethylenedi-, diacetates and manufacture and industrial/professional use are highly controlled and occupational measurements of exposure to workers are available limiting the possibility of exposures.

 

Based on the available information, Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.