Bis(2-dimethylaminoethyl)(methyl)amine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Febr. 20, 2012 to Febr. 6, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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

Details on test material:

- Name of test material (as cited in study report): Pentamethyldiethylentriamine
- Substance type: Nonaromatic amine
- Physical state: Liquid
- Analytical purity: 98.4% (w/w)
- Impurities (identity and concentrations): water 0.17% (w/w)
- Batch No.: 60/11
- Expiration date of the batch: 11/2012
- Stability under test conditions: stable
- Storage condition of test material: Test material was stored in ventilable room in original packaging.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Koleč u Kladna, Czech Republic, RČH CZ 21760152
- Age at study initiation: 10 weeks – on arrival
- Weight at study initiation: cca 325 g (males), cca 225 g (females)
- Selection of animals: random selection according to the internal rule – at the beginning of the study the weight variation of animals in groups of each sex should not exceed ± 20% of the mean weight
- Fasting period before study: no
- Housing: individually ventilated cages (IVC) – special animal room – 2 rats of the same sex in one cage in pre-mating period, during mating period – one male and one female in one cage, pregnant females – individually, offspring – with mother, satellite animals - 2 rats of the same sex in one cage
- Diet: complete pelleted diet for rats and mice in SPF breeding (ST BERGMAN, manufacturer: Ing Miroslav Mrkvička – Výroba krmných směsí, Mlýn Kocanda 19, Jesenice u Prahy, Czech Republic). Diet was sterilised before using and was analysed for nutrients (once a year) and bacteriologically examined (every two months) on a regular basis.
- Water: drinking water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3°C
- Relative humidity: 30-70%
- Bedding: sterilized soft wood fibres
- Photoperiod: 12 hour light / 12 hour dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

The test substance solution was administered to the stomach by gavage. The animals were treated 7 days per week at the same time. The vehicle control group was administered by aqua pro injectione in the same volume.

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighted into glass beaker and the beaker was replenished by aqua pro injectione. The solution was mixed by magnetic stirrer (600 rpm) for 10 minutes and then it was mixed continually during administration. The concentrations of solutions at all dose levels were adjusted to ensure the administration of 1 mL per 100 g of body weight. The application form was prepared daily just before administration.

Details on mating procedure:

Animals were mated from the 15th day of study to 28th day. Mating 1 : 1 (one male to one female in one cage) was used in this study. Each morning the females were examined for presence of spermatozoa in vaginal smears. Day 0 of pregnancy was defined as the day the sperms were found. Pregnant females were housed individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verification of test substance concentration in vehicle was performed before the study when the stability and the homogeneity of application form were examined. From the results of analyses (homogeneity and stability) followed that the solution of the test substance in vehicle prepared at defined laboratory conditions (laboratory temperature, preparation of solution by defined manner, continual mixing) was homogenous and stable at least for 120 minutes starting with preparation of the application form.

The application form for analysis was prepared in the same manner as for application to animals. The test substance was weighted into volumetric flask and was replenished by the vehicle. The solution was mixed by magnetic stirrer (600 rpm) for 10 minutes. Two concentrations of application form were prepared (5 mg/10 mL and 500 mg/10 mL).

The stability of the application form
The stability of the application form was checked by analyses of the application form within 120 min (at the time 0 min, 30 min, 60 min and 120 min). The interval 0 min represents the time after 10 minutes of mixing by magnetic stirrer (600 rpm) for both concentration levels.

The homogeneity of the application form
The homogeneity of the application form was checked by determination of a concentration of the test substance in three places of solution (at the bottom, in the middle and at the surface).

The determination of the test substance was performed by gas chromatography with FID detector.

Duration of treatment / exposure:

According to the guideline, the study was performed as the combined repeated dose toxicity study with the reproduction / developmental toxicity screening test.
The duration of treatment:
Parental males: 42 days [1st day – 14th day (pre-mating) → 28th day (mating) → 42nd day of study]
Parental females: [1st day – 14th day (pre-mating) → 28th day (mating) → gestation → lactation → day 4 post partum]
Non-pregnant females (without evidence of copulation):
[1st day – 14th day (pre-mating) → 28th day (mating) → 54th day of study]
Non-pregnant females (with evidence of copulation):
[1st day – 14th day (pre-mating) → 28th day (mating) → 25th day after confirmed mating (max. 54th day of study)]

Frequency of treatment:

7 days per week at the same time

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 3

Remarks:

Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 4

Remarks:

Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females and 12 males per group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The dose levels for study were determined on the basis of results of a dose-range finding study (DRFS)
DRFS: 14 days, dose levels 50, 100, 200 and 400 mg/kg bw/day

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

HEALTH CONDITION CONTROL
All rats were observed pre-experimentally to ensure that only the animals exhibiting normal behavioural activity would be entered into the study. During the administration period they were examined for behaviour changes each day before, during application and immediately after application.

MORTALITY
Twice daily

CLINICAL OBSERVATION
Males and Females
All rats were observed daily during the administration period.
This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day – at the time of expectation of maximal effect of the test substance. Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATION
This observation was carried out before the first application and then weekly. At the first part of observation the behaviour of animals in the cage was monitored: piloerection, posture, position of eyelids, breathing, tonic or clonic movements, stereotypes or bizarre behaviour.
The second part was the observation during the removal from cage: reaction to handling, elasticity of skin, colour of mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT
The body weight of animals was recorded on automatic balances with group mean computing module on specified days:
males - weekly
females - weekly in pre-mating and mating period, during pregnancy 0., 7th, 14th, 20th day, during lactation 0. or 1st, 3rd and 4th day
All animals were weighed immediately before euthanasia too.
Weight increment was computed as a mean per group (in grams).

FOOD CONSUMPTION
In a specified day the remainder of pellets was weighed in each cage, the new food was weighed out and the food consumption for the previous week was computed.
In males mean values were calculated for each week of the study (except the mating period). Food consumption for animal/day was calculated from mean values of each group. The same way of calculation of mean food consumption was used for females in pre-mating period. In pregnancy and lactation period mean individual values (grams/animal/day) were calculated for each week of the study. Mean food consumption for each group was calculated from individual values. Non-pregnant females (females without parturition) were not included in calculation of mean food consumption in pregnancy and lactation period.

WATER CONSUMPTION
No

Sperm parameters (parental animals):

Parameters examined in parental males: sperm motility, sperm morphology
Sperm motility
Sperm samples were taken from one epididymis and sperm motility was assessed from these samples. The motility of sperm was determined by microscopic examination of the prepared sperm suspension.
Sperm morphology
Sperm samples were taken from one epididymis and sperm morphology was assessed from these samples. A smear from the sperm suspension was prepared and stained (Giemsa staining). The morphology of sperm was determined by microscopic examination.

Litter observations:

All pups were observed in natural conditions in their cages daily during the lactation. Changes in behavioural abnormalities were recorded. Detailed examination of each litter was performed as soon as possible after delivery (day 0 or 1 post-partum) and the 4th day of lactation. The number and sex of pups, stillbirths, live births and presence of gross anomalies were recorded.

Postmortem examinations (parental animals):

SACRIFICE
Parental males: 43th day of study
Parental females: 4th day of lactation
Non-pregnant females: 55th day of study or 26th day after confirming mating

GROSS NECROPSY
During the necropsy a revision of the external surface of the body, of all orifices and the cranial, thoracic and abdominal cavities were carried out.

HISTOPATHOLOGY
Organs for histopathological examination were taken out and stored in containers with fixative (buffer 4% formaldehyde). Testes and epididymides were fixed in modified Davidson’s fixative.
Organs for histopathology: pituitary gland, ovaries, uterus, cervix of uterus, vagina, epididymis/epididymides, prostate gland, seminal vesicles and coagulating gland, testes, all gross lesions.

ORGAN WEIGHTS
- males - the absolute weights of testes, epididymis, prostate gland and pituitary gland
- females - absolute weight of ovaries, uterus and pituitary gland
- the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight

Statistics:

The ANOVA test - Analysis of Variance (QC.Expert 2.5) at significance level 0.05 was used for the statistical analysis (the raw data were used for statistical analysis). This statistical analysis was used for the results of body weight, biometry of organs and selected reproduction parameters – number of live born pups, number of corpora lutea, number of implantations, mean weight of pup on the 0./1st day and mean weight of pup on the 4th day. Males/females from control group were compared with males/females from three treated groups.

Reproductive indices:

Male mating index= (number of males with confirmed mating / number of males cohabited) x 100
Female mating index = (number of sperm-positive females / number of females cohabited) x 100
Male fertility index = (number of males impregnating a females / number of males cohabited) x 100
Female fertility index = (number of pregnant females / number of sperm-positive females) x 100
Gestation index = (number of females with live born pups / number of pregnant females) x 100
Survival index = (number of live pups on day 4 post partum* / number of pups born alive+) x 100

* without still born pups (dead pups with anaerial lungs)
+ with dead pups with aerial lungs

Offspring viability indices:

Pre-implantation loss Number of corpora lutea – number of implantations
Post-implantation loss Number of implantations – number of live births
Post-natal loss Number of live births – number of alive at postnatal day 4

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

for details see below

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Details on results (P0)

MORTALITY
There were no unscheduled deaths during the whole study.

HEALTH CONDITION CONTROL
Parental males
In treated males of the lowest and the middle dose level only sporadically insignificantly changes of health condition before, during and immediately after application of the test substance occurred. In males of the highest dose level the following changes were noted: piloerection from the 3rd to the 6th week of the study and disquiet after application from the 3rd to the 5th week of study.
Parental females
In all exposed females of the highest dose level the following changes were detected: piloerection from the 2nd to the 8th week of the study, disquiet after application from the 4th to the 5th week of study and apathy from the 7th to the 8th week of study. In the 8th week salivation after application of the test substance were observed in all exposed females.

CLINICAL SIGNS
Parental males
In all males of the highest dose level salivation was recorded in the 6th week of study.
Parental females
Only piloerection in one female at the highest dose level was observed in the 8th week of study.

BODY WEIGHT AND BODY WEIGHT INCREMENT
Parental males
In males of the middle and the highest dose level decreased body weight was recorded in comparison with control. This difference was dependent on dose level and individual values were extremely variable.
Parental females
- Pre-mating, mating period
The mean body weight increments of the control females and females at the lowest and the middle dose level were well balanced whilst weight increments of females of the highest dose level slightly decreased in the pre-mating period.
- Pregnancy
The body weight increments of mothers at the middle and highest dose levels were decreased than in control group.
- Lactation
Only mothers (females with live pups born) were included in the evaluation of body weight increments during lactation period.
The mean body weight increments of mothers at the middle and the highest dose levels were decreased against control (with statistical significance at the highest dose level).

FOOD CONSUMPTION
Parental males
The mean food consumption of males of the highest dose level was decreased from the 2nd to the 6th week.
Parental females
- Pre-mating period
The mean food consumption of females at all dose levels was balanced with control females in the 1st week of pre-mating period. In the 2nd week the food consumption of females of the highest dose level was decreased.
- Pregnancy
Dose-dependent effect (decrease) manifested till 0-7 days of pregnancy than the mean food consumption of mothers at all dose levels was analogous to control mothers.
- Lactation
The mean food consumptions of mothers of the lowest dose level were slightly decreased whilst the mean food consumption of mothers at the middle and highest dose level were markedly lower than in control females.

REPRODUCTIVE PERFORMANCE
Reproduction Parameters
Treated females of all dose levels were mated. Evidence of copulation was not found in one female at the lowest dose level and in one female at the highest dose level.
The number of females achieving pregnancy was slightly decreased in treated females. Abortion occurred in one female of the middle dose level. The duration of mating of females of all dose levels it was longer against control. The duration of pregnancy of females at the lowest and middle dose levels was similar to the control females. At the highest dose level the duration of pregnancy was shorter against the control group.
The number of females bearing live pups and females with live pups at day 4 after parturition in females at all dose levels was slightly decreased compared to control.
The numbers of corpora lutea and implantations in females of all dose levels were similar to the control.
The numbers of live pups at birth and at day 4 after parturition in females at the lowest and middle dose levels were conformable to the control group. At the highest dose level these numbers were decreased against control.
No significant differences of mating indexes were observed. Fertility indexes were slightly decreased at middle dose level. At the highest dose level decreased survival index was also detected. Gestation indexes of treated groups were analogous to control.
Pre-implantation losses were similar to the control group. Markedly increased post-implantation and slightly increased post-natal losses were recorded at the highest dose level.

HISTOPATHOLOGY
Histopathological examination was performed for the control group and the highest dose level.
Parental males
No histopathological changes were detected in testes, epididymis, and seminal vesicles.
Parental females
No histological changes were detected in ovaries, uterus, vagina and pituitary gland. In reproductive organs only the changes related to previous pregnancy were found.

Results of histopathological examination of other organs of parental males and femals are discussed and stated in the endpoint "Repeated dose toxicity oral" - section 7.5.1 of this IUCLID data set (Repeated dose toxicity part of combined study).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

> 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

for details see below

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

for details see below

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Number and Sex Ratio of Pups
The statistical evaluation of the number of live born pups/per litter, number of corpora lutea and number of implantations was performed. No statistically significant intergroup differences were recorded.
The total number of live pups and mean number of pups per litter at the dose level 300 mg/kg/day was markedly decreased in comparison with the control. The total number of live pups and mean number of pups per litter at the dose levels 100 mg/kg/day was slightly lowest (total number) or similar (mean number of pups). The total number of live pups and mean number of pups per litter at the dose levels 30 mg/kg/day was similar or higher than control. The presence of stillborn pups was recorded only at the dose level 300 mg/kg/day.
In sex ratio no significant differences were recorded in treated groups.

DEVELOPMENT OF PUPS
Presence of stillborn pups was recorded only at the highest dose level.
Mortality of pups in lactation period was detected at the highest dose level. At the middle dose level only one death pup was found.
No differences in development of pups were observed at the control and treated groups.

BODY WEIGHT (OFFSPRING)
The statistical evaluation of mean weight of pup on the 0./1st day and mean weight of pup on the 4th day was performed. No statistically significant intergroup differences were recorded.
Mean body weights of litters at the dose level 100 mg/kg/day were slightly decreased compared to control. Mean weight of the litter at the dose level 300 mg/kg/day was markedly decreased against control. Mean weights of pup recorded at the 1st check of litter after parturition in treated groups were decreased than in control group. Mean body weight increment of pup (from the 1st check of litter after parturition to the 4th day of lactation) was similar in the treated and control groups.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination was performed in all pups. Pathological findings were observed only in stillborn pups: lungs without air, empty stomach. In other examined pups of control and treated groups no pathological findings were recorded.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The value was established mainly on the basis of changes in post-implantation and post-natal development of pups. The increase post-implantation losses could be influenced by maternal toxicity.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established higher than at 300 mg/kg body weight/day.
The NOAEL (No Observed Adverse Effect Level) for the DEVELOPMENT of pups was established at 100 mg/kg body weight/day. The value was established mainly on the basis of changes in post-implantation and post-natal losses. The increase post-implantation losses could be influenced by maternal toxicity.

Executive summary:

Introduction

The test substance, Pentamethyldiethylentriamine, was tested for reproduction/developmental toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, Adopted by the Council on March 22nd 1996.

 

Methods

Wistar rats of SPF quality were used for testing. The test substance was administered in the form of solution in water for injection. Oral application by stomach tube was performed daily. The study included four groups. Each group consisted of 12 males and 12 females. Main groups contained 3 treated groups (doses 30, 100, 300 mg/kg of body weight /day) and one control group (vehicle only). The dose levels for study were determined on the basis of results of a dose-range finding experiment.

The treated groups were administered daily for the following periods:

males and females - 2 weeks prior to the mating period and during the mating period,

pregnant females - during pregnancy and till the 3rd day of lactation,

males - after mating period - totally for 42 days,

non-pregnant females (mated females without parturition) - for 25 days after the confirmed mating.

After the end of administration period the animals of main groups were sacrificed.   

During the study clinical observation and health status control were performed daily. The body weight and food consumption were measured weekly or in the specified time intervals. Vaginal smears were prepared daily during the mating period (until the presence of spermatozoa). Reproduction parameters relevant to pups (number of pups, weight of litters, sex or vitality) were also recorded.

The study was finished by gross necropsy of animals. In all males of all groups the sperm parameters, sperm motility and sperm morphology were examined. The selected organs from parental animals were removed for weighing and histopathological examination.

 

Results

The oral administration of Pentamethyldiethylentriamine to rats by gavage, the dose levels 30, 100 and 300 mg/kg/day, did not cause mortality.

The course of mating, pregnancy and lactation of parental animals, number of females achieving pregnancy, spermiogenesis and sperm parameters, biometry of reproductive organs and pituitary gland, macroscopical and microscopical structure of reproductive organs and pituitary gland of parental animals, sex ratio and development of pups were not adversely affected by the test substance treatment. The slight intergroup differences were considered to be of no toxicological significance.

Male ability to produce sperm that can fertilise eggs and female ability to achieve pregnancy was not significantly changed - number of females achieving pregnancy was similar in control and treated groups. The total number of live pups and mean number of pups per litter were decreased in high-dose females. Presence of stillborn pups was recorded only at the highest dose level. Post-implantation losses were increased in females of the highest dose level (decreased number of life born pups) - the test substance probably had negative influence on early prenatal development of organism in uterus. Evaluation of pup weight revealed adverse effect on intrauterine pup growth attributable to test substance: mean litter weight and mean pup weight at birth was decreased at the highest dose level. The early prenatal growth of pups was influenced by toxic effects in the mothers. The body weight increments of pups at the treated groups from the birth to the 4th day after parturition were similar to the weight increment of pups in control group. The test substance had no negative effect to the postnatal growth of pups.

 

Conclusion

The NOAEL (No Observed Adverse Effect Level) for the REPRODUCTION was established higher than at 300 mg/kg body weight/day.     

The NOAEL (No Observed Adverse Effect Level) for the DEVELOPMENT of pups was established at 100 mg/kg body weight/day. The value was established mainly on the basis of changes in post-implantation and post-natal development of pups. The increase post-implantation losses could be influenced by maternal toxicity.