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Toxicological information

Toxicity to reproduction

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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to the OECD guideline 422 and GLP compliant
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N-(3-aminopropyl)iminodiethanol
EC Number:
225-642-0
EC Name:
N-(3-aminopropyl)iminodiethanol
Cas Number:
4985-85-7
Molecular formula:
C7H18N2O2
IUPAC Name:
2-[(3-aminopropyl)(2-hydroxyethyl)amino]ethan-1-ol
Constituent 2
Reference substance name:
N-(3-aminopropyl)diethanolamine
IUPAC Name:
N-(3-aminopropyl)diethanolamine
Test material form:
other: Clear slightly yellow liquid
Details on test material:
-Identification: N-(3-aminopropyl)diethanolamine (APDEA)
-Molecular formula: C7H18N2O2
-Molecular weight: 162.23
-CAS Number:4985-85-7
-Description: Clear slightly yellow liquid
-Batch: TG70L02N
-Purity: 90.7%
-Test substance storage: At room temperature in the dark
-Stability under storage conditions: Stable
-Expiry date: 30 June 2012
-Conversion factor: The purity of the test substance was 90.7%. Accordingly, a correction factor of 1.1025 was applied to adjust for the purity of the test substance
-Specific Gravity: 1.07
-pH: 12
-Stability in water: Stability for at least 6 hours at room temperature is confirmed over the concentration range 10 to 100 mg/mL, NOTOX Project 496700.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation:
- Fasting period before study:
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete study period.
Mating: Repro females were caged together with Main males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-mating: Main males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Repro females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Lactation: Pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm).

- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days prior to start of treatment.

In the period from 30 June 2011 to 05 July 2011 (10:43) , temperature and relative humidity were not recorded by the REES Centron Environmental Monitoring system. During this period, an alternative (non-GLP) recording system was available, which suggested that the temperature of the animal room was within protocol specifications during that respective period, but relative humidity of was outside protocol specifications.
Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of pupillary reflex tests in the room.
Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 22.0°C
- Humidity (%): 47 - 86%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Elix, Millipore S.A.S., Molsheim, France)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for purity (90.7%) and specific gravity (1.07) of the test substance. The pH value was determined on the first day of dosing for each formulation.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at NOTOX.
- Storage conditions: At ambient temperature.
- Dose volume: 10 mL/kg body weight.
Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: maximum of 14 days
- Proof of pregnancy: vaginal plug day 0 of pregnancy
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually housed in Macrolon plastic cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses by UPLC-MS were conducted on a single occasion during the treatment phase (18 July 2011), according to a validated method (NOTOX Project 496695). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).

In addition, samples taken at the middle position of the container from dosing solutions of Weeks 1, 3 and 5 of the study were taken and stored at =-15°C for possible future analysis. Any remaining samples will be discarded after approval by the sponsor, or at finalization of the study report.

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was = 10%.
Duration of treatment / exposure:
The Repro females were exposed for 43-57 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Repro females 116 and 117 (Group 2) were not dosed on one day (Day 1 of lactation) as they were littering at the moment of dosing.
Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/d
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of a 14-day dose range finding study (NOTOX Project 496700), the dose levels for this combined 28-day oral gavage study with reproduction/developmental toxicity screening test were selected to be 100, 300 and 1000 mg/kg.
- Rationale for animal assignment (if not random): computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated Repro females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean diet consumption calculated as g food/kg body weight/day: Yes, weekly, except for Main males and Repro females which were housed together for mating and for Repro females without evidence of mating. Food consumption of mated Repro females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation

WATER CONSUMPTION: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Oestrous cyclicity (parental animals):
No
Sperm parameters (parental animals):
No
Litter observations:
STANDARDISATION OF LITTERS
No

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
-Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were
evaluated.
-Clinical signs: At least once daily, detailed clinical observations were made for all animals.
-Body weights: Live pups were weighed on Days 1 and 4 of lactation.
-Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals
- Maternal animals: All surviving animals; Repro females which delivered: Lactation Days 5-7. Repro females which failed to deliver: Post-coitum Days 25-28 (females 102, 106, 118 and 119; with evidence of mating) or approximately 21 days after the last day of the mating period (female 101; without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: All animals were subjected to macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. Descriptions of all macroscopic abnormalities were recorded.
The numbers of former implantation sites and corpora lutea were recorded for all paired Repro females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table below were prepared for microscopic examination and weighed, respectively.

Microscopic observation
Identification marks: not processed
Adrenal glands
All gross lesions
Brain - cerebellum, mid-brain, cortex
Cervix
Clitoral gland
Heart Thyroid including parathyroid if detectable
Kidneys
Liver
Lung, infused with formalin
Ovaries
Pituitary gland
Spleen
Thymus
Uterus
Vagina
Organs weighted
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Lungs
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid including parathyroid
Uterus (including cervix)

Organs weighted
Adrenal glands
Brain
Epididymides
Heart
Kidneys
Liver
Lungs
Ovaries
Prostate
Seminal vesicles including coagulating glands
Spleen
Testes
Thymus
Thyroid including parathyroid
Uterus (including cervix)
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed by decapitation between Days 5-7 of lactation.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of: All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated.

HISTOPATHOLOGY / ORGAN WEIGTHS
No
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Reproductive indices:
Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded. Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of pregnant females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

Mating index (%) = (Number of females mated / Number of females paired) x 100
Fertility index (%) = Number of pregnant females / Number of females paired) x 100
Conception index (%) = Number of pregant females / number of females mated) x 100
Offspring viability indices:
Gestation index (%) = Number of females bearing live pups / Number of pregnant females) x 100
Duration of gestation = Number of days between confirmation of mating and the beginning of parturition
Percentage live males at First Litter Check = (Number of live male pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check = (Number of live female pups at First Litter Check / Number of live pups at First Litter Check) x 100
Percentage of postnatal loss (Days 0-4 of lactation) = (Number of dead pups on Day 4 of lactation / Number of live pups at First Litter Check) x 100
Viability inde = (umber of live pups on Day 4 post partum / Number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

See RSS in section 7.5.1 Repeated dose toxicity: oral

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Fertility
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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)
One pup of the control and low dose groups and two pups of the mid and high dose groups were found dead or missing during the first days of lactation. Pups missing were most likely cannibalised.
No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS (OFFSPRING)
Incidental clinical symptoms of pups consisted of pale appearance (one pup in high dose group) and insufficient milk in the stomach (one pup each in the low and mid dose groups). The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT (OFFSPRING)
Body weights of pups were considered to have been unaffected by treatment.

GROSS PATHOLOGY (OFFSPRING)
One pup in the mid dose group found dead at first litter check showed absence of milk in the stomach.
No other findings were noted. The nature and incidence of this finding remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Gestation

Gestation index and duration of gestation were unaffected by treatment up to 1000 mg/kg.

The statistical significant increase noted for duration of gestation at 1000 mg/kg was not considered toxicologically relevant as all values were within normal limits (all individual values were either 21 or 22 days).

Parturition/maternal care

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development

Number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Applicant's summary and conclusion

Conclusions:
In a study performed according to the OECD 422, N-(3-aminopropyl)diethanolamine was administered by daily oral gavage followed by a 14-day recovery period to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day. Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg was established. A reproduction and developmental NOAEL of at least 1000 mg/kg/day was derived.
Executive summary:

In a study performed according to the OECD 422, N-(3-aminopropyl)diethanolamine was administered by daily oral gavage followed by a 14-day recovery period to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/day (de Raaf-Beekhuijzen, 2011). Main and Recovery animals were exposed for 29-32 days, i.e. 2 weeks prior to mating, and during the mating period for Main males. The Repro females were exposed for 43-57 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Formulation analysis showed that the formulations were prepared accurately and homogenously.

At 1000 mg/kg, parental toxicity consisted of reduced locomotor activity (total movements) with a normal habituation pattern, reduced body weight gain for male animals, affected haematological parameters (decreased values for monocytes, neutrophils, red blood cells, haemoglobin, haematocrit, and mean corpuscular volume, and increased prothrombin time, white blood cells and lymphocytes), changes in clinical biochemistry parameters (increased values for aspartate aminotransferase, alanine aminotransferase, urea, calcium, potassium, total bilirubin and bile acids, and decreased concentration of total protein and albumin), and organ weight changes (increased liver, kidneys and adrenals weights and decreased thymus weights). No other toxicologically relevant findings that indicated a sign of systemic toxicity was noted. No macroscopic and microscopic lesions were noted for systemic toxicity. The toxicological relevance of organ weight changes is uncertain without accompanying microscopic findings. The only apparent treatment related macroscopic and microscopic changes was in the stomach that is probably related to irritancy of the test substance.

They were several dark red or reddish foci at the glandular mucosa of the stomach at macroscopic examination, and microscopic stomach abnormalities consisting of acute inflammation, hyperplasia, vacuolative degeneration, necrosis and haemorrhage.

Most findings recovered during the 14-day treatment free period. Acute inflammation persisted in two male and one female animals. Even though increased liver and kidneys weight were noted after the recovery period, it was not considered to be toxicologically relevant without any accompanying clinical chemistry parameters and microscopic findings.

In addition, increased absolute and/or relative kidneys weights were noted for males and females at 300 mg/kg and for Repro females at 100 mg/kg. Only for the Repro females at 300 mg/kg, these values were just outside the historical control data. In the absence of corroborative changes in clinical biochemistry parameters and histopathology, and lack of any toxicity at these doses, the kidneys weight changes were not considered adverse.

Minimum hyperplasia of non-glandular epithelium at the stomach were noted for a few animals treated at 300 mg/kg and one male animal each in the control and 100 mg/kg group. However, it should be noted that thickening (hyperplasia) of the non-glandular epithelium in the region of the limiting ridge is a subjective opinion and is often confounded/obfuscated by tangential sectioning. Both hyperplasia and vacuolative degeneration of the non-glandular epithelium in the area of the limiting ridge can be observed in normal control animals (common findings) and are likely to be less toxicologically relevant at lower dose levels where concomitant inflammation was not present.

At 300 mg/kg, slightly increased calcium levels were noted. In the absence of any corroborative findings and as the change was very slight, this finding was not considered toxicologically relevant. No toxicologically significant changes were noted in any of the remaining parental parameters investigated in this study (i.e. clinical appearance, functional observations and food consumption).

No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg).

No toxicologically significant changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites).

No developmental toxicity was observed up to the highest dose level tested (1000 mg/kg).

No toxicologically significant changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy).

In conclusion, treatment with APDEA by oral gavage followed by a 14-day recovery period in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg body weight/day revealed parental toxicity at 1000 mg/kg body weight/day. No reproduction and developmental toxicity was observed for treatment up to 1000 mg/kg body weight/day.

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg was established. A reproduction and developmental NOAEL of at least 1000 mg/kg/day was derived.

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