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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2aug1993 / 12jan1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

EC Number:

219-145-8

EC Name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Cas Number:

2372-82-9

Molecular formula:

C18H41N3

IUPAC Name:

N-(3-aminopropyl)-N-dodecylpropane-1,3-diamine

Details on test material:

- Name of test material (as cited in study report): P4150
- Physical state: colourless liquid
- Analytical purity: 30.2% aqueous solution
- Lot/batch No.: PN-93-12
- Storage condition of test material: in the dark at ambient temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca. 245 g (males), ca. 9 weeks (females)
- Fasting period before study:
- Housing: females: individually in polypropylene cages with stainless steel grid bottoms and mesh tops, measuring 42 x 27 x 20 cm. Prior to mating females were housed in pairs in this type of cage. Males: singly in cages of similar design, measuring 58 x 38.5 x 20 cm.
- Diet : Rat and Mouse Breeder Diet No. 3 (Expanded) SQC, ad libitum
- Water: domestic mains water, ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Fresh solutions were prepared daily. For high concentration group, the requisite quantity of the test material was weighed into a labelled container. The necessary volume of vehicle was added, and mixing was by inversion, as required. For lower concentrations aliquots of the high dose solution were diluted with the vehicle to give required concentrations.

VEHICLE

- Amount of vehicle (if gavage): 10 mL/kg bw
- Concentration in vehicle: 2.5, 7.7, 20 mg/mL (calculated for pure substance)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A single 10 mL sample was taken from each concentration on the 1st and 8th day of dosing and triplicate sub-samples were analyzed for concentration and homogeneity in the IRI Analytical Chemistry Laboratory by method # 7034.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: continuous until mating was detected
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0

Duration of treatment / exposure:

Gestations Day 6-16

Frequency of treatment:

daily

Duration of test:

Until Day 20 of gestation

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 25, 75, 200 mg/kg bw/day (test substance as 30.2% aqueous solution)
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 7.5, 22.5 and 60 mg/kg bw/day (pure substance)
Basis:
actual ingested

No. of animals per sex per dose:

25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on a separate dose range finding study
- Rationale for animal assignment (if not random): computer generated series of randomly sequenced numbers, ensuring that siblings and females mated by the same male were not placed in the same treatment group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily. In addition animals were checked for viability at the beginning of each day and as late as possible on each day.

BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 9, 13, 17 and 20 of gestation

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily, commencing on Day 3 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: the contents of thoracic and abdominal cavity

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, all per litter
- Skeletal examinations: Yes, half per litter
- Head examinations: No

Statistics:

Weight gain was analysed using the Kruskal-Wallis test, with comparisons with the control using a modified Dunn's procedure. Food consumption was analysed by parametric analysis of variance, with homogeneity of variance checked using Levene's test and normality of data by the Shapiro-Wilk test. Comparisons with the control were made using Dunnett's t-test. For all other parameters, interpretation was based on examination of the individual and group mean values.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All animals at 200 mg/kg/day test material (corresponding to 60 mg/kg bw/day pure substance) and many at 75 mg/kg/day (corresponding to 22.5 mg/kg bw/day pure subtance) showed reaction to treatment. The principle findings were dyspnoea, salivation with associated brown staining, piloerection and indications of reduced activity (such as hunched, ataxic and cold to touch). Two animals at 200 mg/kg/day were killed prematurely: necropsy findings included intestinal distension, and therefore the similar distension observed for a further animal was also considered to be associated with treatment. The other clinical observations and necropsy findings were considered to be either associated with the above findings or to be incidental.
There was a dose related reduction in weight gain over the dosing period at 75 and 200 mg/kg/day (corresponding to 22.5 and 60 mg/kg bw/day pure substance), which had become apparent by Day 9 of gestation. At 200 mg/kg/day, weight gain over Days 6-17 was significantly lower than control (P<0.0 1) and 5 animals at this level showed weight loss between Days 9 and 13 of gestation. Gain over Days 6-17 at 75 mg/kg/day, although lower than control, was not statistically significant (P> 0.05).
Body weight performance at 25 mg/kg bw/day (corresponding to 7.5 mg/kg bw/day) was essentially similar to control.
There was a reduction in food consumption at 200 mg/kg/day, which persisted throughout the dosing period. At 75 mg/kg/day, food consumption was marginally reduced. At both these levels, food consumption over the dosing period was significantly lower than control (P<0.01).
Food consumption at 25 mg/kg/day was similar to Control.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The mean number of implants at 25 and 75 mg/kg/day (corresponding to 7.5 and 22.5 mg/kg bw/day pure substance) was slightly lower than control or at 200 mg/kg/day (corresponding to 60 mg/kg bw/day pure substance). However, this could not be attributed to treatment because the number of implants would have been established prior to commencement of treatment.
The incidence of early embryonic deaths, and hence of total dead implants was greater at 200 mg/kg/day (60 mg/kg bw/day pure substance) than in control. This increase was largely associated with three animals which had large numbers of early deaths. The incidence of dead implants at 25 and 75 mg/kg/day (7.5 and 22.5 mg/kg bw/day pure substance) was similar to control.
Mean foetal weight at 200 mg/kg/day (60 mg/kg bw/day pure substance) was slightly lower than control, with the difference approaching but not achieving statistical significance(P>0.05).
The number and type of major foetal abnormalities were considered to be typical of those seen in historical data. Taken together, the incidences of minor abnormalities and variants were typical of those that would be expected from historical data and were similar in all groups. The group values indicating the state of skeletal ossification were similar in all groups. Slight differences were considered too small to be attributed to treatment.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study no teratogenic effects (as indicated by the incidences of foetal malformations, anomalies and variants) were found up to 200 mg P4150/kg/day (60 mg a.i./kg/day) of the test substance.

Executive summary:

Based on Guidelines: OECD 414 and EPA-FIFRA 83-3 100 mated female and 60 male rats were used for the study. After mating females were housed singly with separate food and water for each cage. Batches of diet and water were analysed throughout the study. Pairing was on the basis of one male to two females. For each female cohabitation with a male was continuous until mating was detected. A vaginal lavage was examined each morning and the day of detection of sperm in the lavage, or of a copulatory plug in situ was considered to be Day 0 of gestation. On detection of mating, females were randomly assigned to treatment groups. The treatment groups were arranged as follows: Group no. Treatment/dose level mg P4150/kg/day Animal nos. 1 Control 0 1-25 2 Low dose 25 26-50 3 Intermediate 75 51-75 4 High 200 76_100,160* *Replacement animal following death of animal no. 86 Dose levels were determined from the results of a separate dose finding study in rats. In the study, very slight maternal toxicity was demonstrated in mated animals at 100 and 150 mg/kg/day. However, unequivocal toxicity was seen in a supplementary group of unmated animals at 200 mg/kg/day. There was no obvious effect on pregnancy performance or foetal weight in the mated animals. Females were dosed orally by gavage at a volume of 10 ml dosing solution per kg of body weight using a steel dosing cannula. The volume of solution to be administered each day was determined each day by the weight of the animal as measured at the time of administration. The animals were dosed once daily at approximately the same time each day over Days 6— 16 inclusive of gestation. The test material was formulated as a solution in water. Fresh solutions were prepared daily. A 10 ml sample from each concentration was taken on the first and eight day of dosing for analysis for concentration, homogeneity and stability. Clinical observations were recorded for reaction to treatment each day. In addition, all animals were checked for viability at the beginning and end of each day. Individual body weights were recorded on Days 0, 6, 9, 13, 17 and 20 of gestation. The weight of food consumed by each mated female was recorded daily, commencing on Day 3 of gestation. Females were killed by carbon dioxide asphyxiation. Foetuses were killed by chilling at ca 4°C for ca 5 mm before fixation. Animals that died prematurely during the study were examined at necropsy and included examination of the cranial contents. Other necropsies were conducted on Day 20 of gestation but did not include cranial examination. The thoracic and abdominal cavities were inspected, and any lesions were described, samples were taken where necessary. The reproductive tract was weighed intact, with examination of the uterus contents. The number and position of all implants was recorded. Each implant was classified as being live, a foetal death, i.e. death occurred after day 16 of gestation, or an early embryonic death, i.e. only placental remains or a decidual scar visible. The number of corpora lutea graviditatis in each ovary were recorded. Each live foetus within the litter was individually identified and its weight recorded. The foetuses were examined for externally visible abnormalities. Half the foetuses were fixed in methylated ethyl alcohol and examined for occurrence of gross visceral abnormalities. These foetuses were then cleared in potassium hydroxide and glycerol and the skeletons stained with Alizarin Red S for examination for abnormalities and variants, including state of ossification. The other half of the foetuses were fixed in Bouin’s fluid and examined for visceral abnormalities by a free-hand serial sectioning technique from that of (1965) Teratology: Principles and Techniques, The of . The sex of the foetus was determined during the visceral examination. Statistical analysis was used where appropriate. Weight gain was analysed using the Kruskal-Wallis test, with comparisons with the control using a modified Dunn’s procedure. Food consumption was analysed by parametric analysis of variance, with homogeneity of variance checked using Levene’s test and normality of data by the Shapiro-Wilk test. Comparisons with the Control were made using Dunnett’s t-test. For all other parameters, interpretation was based on examination of the individual and group mean values. Maternal toxicity was indicated at 75 and 200 mg P4150/kg/day by reduced body weight gain and food consumption and by clinical reactions to treatment, which in 2 animals at 200/kg/day required premature sacrifice: dyspnoea, salivation with associated brown staining, piloerection and indications of reduced activity (such as hunched, ataxic and cold to touch).. At 200 mg/kg/day, there was an increased incidence of early embryonic deaths: the 3 females with the greatest number of early deaths were all among the 5 females showing weight loss between Days 9 and 13 of gestation, suggesting that the early deaths may have been secondary to maternal toxicity. Mean foetal weight at this level was slightly lower than Control. At 25 and 75 mg/kg/day, there were no obvious effects on pregnancy performance or foetal weight. The incidences of foetal malformations, anomalies and variants were essentially similar in all groups.