Bis(2-dimethylaminoethyl)(methyl)amine

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

First mating results: October 13, 2015; First administration: October 19, 2015; Termination of the in-life part: November 10, 2015; Termination of skeletal examination March 22, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-dimethylaminoethyl)(methyl)amine

EC Number:

221-201-1

EC Name:

Bis(2-dimethylaminoethyl)(methyl)amine

Cas Number:

3030-47-5

Molecular formula:

C9H23N3

IUPAC Name:

(2-{[2-(dimethylamino)ethyl](methyl)amino}ethyl)dimethylamine

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Pentamethyldiethylenetriamine
- Molecular formula (if other than submission substance):
- Molecular weight (if other than submission substance):
- Smiles notation (if other than submission substance):
- InChl (if other than submission substance):
- Structural formula attached as image file (if other than submission substance): see Fig.
- Substance type:
- Physical state: liquid
- Analytical purity: >98.5% (wt)
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition:
- Purity test date:
- Lot/batch No.: 890051702
- Expiration date of the lot/batch:
- Radiochemical purity (if radiolabelling):
- Specific activity (if radiolabelling):
- Locations of the label (if radiolabelling):
- Expiration date of radiochemical substance (if radiolabelling):
- Stability under test conditions:
- Storage condition of test material: At +10°C to +25°C, to be stored in original packaging in a well-ventilated place.
- Other:

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: on day 0 of pregnancy: 60 days
- Weight at study initiation: on day 0 of pregnancy: 211.1 – 270.1 g
- Fasting period before study: no
- Housing: singly (except during the mating period)
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): 15 to 20 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark 7 12 hrs light
IN-LIFE DATES: From: October 13, 2015 To: November 10, 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations were freshly prepared every day. The test item was diluted in the vehicle to the appropriate concentration and was administered orally at a constant volume once daily from the 6th to the 20th day of gestation.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 10, 30 or 60 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg b.w./day
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analysed according to the analytical method (Titration with HCl, 1 M) for the determination of the test item in liquid
formulation samples validated by LPT.
The measured actual concentrations of the test item-vehicle mixtures were between 97.4% and 99.8% of the nominal concentrations,
indicating correctly prepared formulations, which were stable for at least 24 hours.

Details on mating procedure:

Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen.
Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A postmortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.

Duration of treatment / exposure:

from the 6th and lasting until the 20th day of pregnancy

Frequency of treatment:

once daily

Duration of test:

5 months (October 13, 2015 To: March 22, 2016)

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:
actual ingested
20, 60, 120 mg/kg b.w./day

No. of animals per sex per dose:

25 rats per dose (to obtain 20 litters per dose for evaluation)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: in agreement with the Sponsor based on the results of a dose-range-finding study for a prenatal developmental toxicity study in pregnant rats (LPT study no. 32469).
- Rationale for animal assignment (if not random): The rat is a commonly used rodent species for such embryotoxicity studies.
- Other: no

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.
- Cage side observations checked : behavioural changes, reaction to treatment, or illness
- Viability: early in the morning and again in the afternoon of each working day to look for dead or moribund animals

DETAILED CLINICAL OBSERVATIONS: No
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: on day 0 of gestation followed by daily weighing - always at the same time of the day.
The body weight gain was calculated in intervals (i.e. day 0-3, 3-6, 6-9, 9-12, 12-15, 15-18 and 18-21), for the whole study (gestation day 0 - 21) and for the period after the start of dosing (gestation day 6 to gestation day 21). Furthermore the carcass weight and the net weight change from day 6 is given.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: internal organs and placentae

OTHER:

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
- Other: location of the fetuses in the uterus

Fetal examinations:

- External examinations: Yes: [all per litter] for damages, especially for malformations
- Soft tissue examinations: Yes: [half per litter] for soft tissue anomalies. Body sections were made and examined according to WILSON.
- Skeletal examinations: Yes: [half per litter] skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes: [half per litter ] included in soft tissue examination of body sections
In addition:
(a) Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.
(b) The number of fetuses (alive and dead) and placentae (location in the uterus and the assignment of the fetuses) was determined.
(c) Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spont aneous movement).
(d) Number and size of resorptions were determined.
(e) Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
(f) Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
(g) All fetuses (dead and alive) were inspected externally for damages, especially for malformations .
(h) The fetuses were sacrificed by an ether atmosphere.

Statistics:

Parametrical data:
The statistical evaluation of the parametrical values was done by Provantis using the following settings:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
Non-parametrical data:
The statistical evaluation of non-parametrical values was done using the FISHER (n < 100) or Chi2 test (n ≤ 0.01) (p ≤ 0.05 and p ≤ 0.01).

Indices:

Corpora lutea: number per dam, absolute number per group, mean per group
Implantations: number per dam, distributions in the uterine horns, absolute number per group, mean per group
Resorptions: number per dam, distributions in the uterine horns, absolute number per group, mean per group, early resorptions < 2 mm, late resorptions > 2 mm
Weight of placentae: individual data per fetus, mean per litter, mean per group, mean per sex and group
Weight of fetuses: individual data per fetus (alive and dead), mean per litter, mean per group, mean per sex and group
Fetuses: number per dam (alive), number per dam (dead), number of fetuses (alive + dead) per sex and dam, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, Male/female ratio (alive + dead)
Runts: number per dam, number per group
Malformed fetuses: type of malformation, individual data per fetus, number and incidence (%) per group and litter, Total malformation rate [%] =
malformed fetuses per group / fetuses per group x 100
Fetuses with variations: type of variation, individual data per fetus, number and incidence (%) per group and litter, Total variation rate [%] =
fetuses per group with variations / fetuses per group x 100
Fetuses with retardations: type of retardation, individual data per fetus, number and incidence (%) per group and litter, Total retardation rate [%] =
fetuses per group with retardations / fetuses per group x 100
Pre implantation loss [%]: Corpora lutea - Implantations / Corpora lutea x 100
Post implantation loss [%]: Implantations - living fetuses / Implantations x 100

Historical control data:

Summarized results of the 59 last embryotoxicity studies in performed at LPT in the years 2000 to December 2015 Sprague-Dawley rats (Charles River Deutschland GmbH) (1. General reproductive indices, 2. Skeletal retardations, 3. Variations a) skeletal, b) visceral, c) external 4. Malformations)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: 120 mg/kg: Breathing sounds were noted in 12 of 20 dams on one to six test days, from gestation day 7 onwards until laparotomy on gestation day 21. Reduction in body weight and food consumption.

Details on maternal toxic effects:
Mortality: No test item-related premature death was noted in the control group and in the treatment groups (20, 60 or 120 mg Pentamethyldiethylenetriamine/kg b.w./day).
Clinical signs: At 120 mg Pentamethyldiethylenetriamine/kg b.w./day, breathing sounds were noted in 12 of 20 dams on one to six test days, from gestation day 7 onwards until laparotomy on gestation day 21.

Affected dams Incidence of breathing sounds
(number of test days)
12 of 20 dams between gestation days 7 - 21
nos. 85, 86, 89, 96: 1 test day
nos. 87, 92: 2 test days
no. 90: 3 test days
nos. 79, 80, 83, 91: 5 test days
no. 88: 6 test days

Body weight and body weight gain: At 120 mg Pentamethyldiethylenetriamine/kg b.w./day a slight but statistically significantly (p ≤ 0.05 or 0.01) reuced body weight in comparison to the control group was noted on every day between gestation days 7 and 21 (maximum on gestation day 21 with 6.5% below the value of the control group).Body weight gain for the whole study period was 16.9% (p ≤ 0.01) below the value of the control group for the dams of the high dose group (120 mg Pentamethyldiethylenetriamine/kg b.w./day). The 3-day interval of body weight gain revealed statistically significant (p ≤ 0.01) reductions in body weight gain between gestation days 6 and 9 and gestation days 18 and 21 at the high dose level (120 mg Pentamethyldiethylenetriamine/kg b.w./day).
Food consumption: At 120 mg Pentamethyldiethylenetriamine/kg b.w./day slight but statistically significant (p ≤ 0.05 or p ≤ 0.01) reductions in food consumption were noted on altogether 7 test days (between gestation days 6 and 11 and between gestation days 20 and 21 (max. 20.9% below the values of the control group)).
Drinking water consumption: The visual appraisal of the drinking water consumption revealed no test item-related influence at any tested dose level.
Necropsy findings: No changes were noted during the macroscopic inspection of the dams at necropsy.
Uterus and carcass weights: A statistically significant (p ≤ 0.01) reduction in the carcass weight (7.5% below the value of the control group) was noted at the high dose level (120 mg Pentamethyldiethylenetriamine/kg b.w./day).
Reproduction data: No influence on the reproductive parameter (number of implantation sites, resorptions and fetuses) was noted.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Examination of the fetus: Mortality No dead fetuses were noted in any of the test groups.
Body weight of the fetuses and the placentae: No test item-related changes were noted.
Fetal alterations: Malformations No test item-related malformation was noted during the macroscopic examination at laparotomy (external inspection and inspection of the organs and tissues for gross lesions). The skeletal examination according to DAWSON and the soft tissue examination according to WILSON also revealed no test item-related malformations.
Variations: The macroscopic examination at laparotomy, the skeletal examination according to DAWSON and the soft tissue examination according to WILSON revealed no test item-related variations.
Retardations: No test item related retardation was noted during the skeletal examination according to DAWSON.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: teratogenicity

2

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

3

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 60 mg Pentamethyldiethylenetriamine/kg b.w./day for the dams. No test item-related death was noted.
At 120 mg Pentamethyldiethylenetriamine/kg b.w./day signs of toxicity were noted in the form of a reduced body weight and a transiently reduced food consumption.
The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 120 mg Pentamethyldiethylenetriamine/kg b.w./day.
The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.
No dead fetuses, no test item-related malformations, variations or retardations were noted.

Executive summary:

In this prenatal developmental toxicity study, the test item Pentamethyldiethylenetriamine was administered orally to female rats at dose levels of 20, 60 or 120 mg/kg b.w./day from the 6th to 20th day of pregnancy.

Under the present test conditions, the no-observed-adverse-effect level (NOAEL) was 60 mg Pentamethyldiethylenetriamine/kg b.w./day for the dams.

No test item-related death was noted.

At 120 mg Pentamethyldiethylenetriamine/kg b.w./day signs of toxicity were noted in the form of a reduced body weight and a transiently reduced food consumption.

The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 120 mg Pentamethyldiethylenetriamine/kg b.w./day.

The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item.

No dead fetuses, no test item-related malformations, variations or retardations were noted.