Bis(2-dimethylaminoethyl)(methyl)amine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

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

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

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

Remarks:

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

Remarks:

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

Remarks:

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

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

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

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: 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

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).

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

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

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.

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.

Reproductive effects observed:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimish score 1 (GLP compliance, no deviations)

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Higher-tier fertility study (two-generation study) is not required at this tonnage band, since there were no

adverse effects observed in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) in reproductive organs or tissues. Therefore, there is no data gap in fertility.

Short description of key information:
The key information is based on the results of the study for reproduction/developmental toxicity using the OECD Test Guideline No. 422: Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.
The NOAEL for reproduction was determined: > 300 mg/kg bw/day.

Justification for selection of Effect on fertility via oral route:
Only one study is available.

Effects on developmental toxicity

Description of key information

The key information is based on the results of the prenatal developmental toxicity study using the OECD Test Guideline No. 414 and EC method B.31. The no-observed-adverse-effect level (NOAEL) for the fetal organism was above 120 mg Pentamethyldiethylenetriamine/kg bw/day.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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

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)

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

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)

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.

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

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.

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

> 120 mg/kg bw/day

Basis for effect level:

other: fetotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

120 mg/kg bw/day

Species:

rat

Quality of whole database:

Klimish score 1 (GLP compliance, no deviations)

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

The aim of the study was the examination of the influence of Pentamethyldiethylenetriamine on the pregnant rat and the fetus when administered orally during the critical period of organogenesis and the fetal development (6th to 20th day of gestation).

In this prenatal developmental toxicity study, the test itemPentamethyldiethylenetriaminewas administered orally to female rats at dose levels of 20, 60 or 120 mg/kgbw/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/kgbw/day for the dams.

No test item-related death was noted.

At 120 mg Pentamethyldiethylenetriamine/kgbw/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 bw/day.

Justification for classification or non-classification

Based on the data available ( combined repeated dose toxicity study with the reproduction/ developmental toxicity screening test according to OECD Test Guideline No. 422 and prenatal developmental toxicity study according to OECD Test Guideline No. 414 and EC method B.31), the substance is not classified according to CLP Regulation (Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures).

No adverse effects were observed in the combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) on reproductive organs or tissues. The NOAEL (No Observed Adverse Effect Level) for the reproduction was established to be higher than 300 mg/kg body weight/day.

In prenatal developmental toxicity study the no-observed-adverse-effect level (NOAEL) for the fetal organism was above the highest tested dose level - 120 mg Pentamethyldiethylenetriamine/kg bw/day. The reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item and no dead fetuses, no test item-related malformations, variations or retardations were noted.