N-(3-aminopropyl)iminodiethanol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to the OECD guideline 422 and GLP compliant

Reason / purpose for cross-reference:

reference to same study

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

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.

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.

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

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

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: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

See RSS in section 7.5.1 Repeated dose toxicity: oral

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'

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

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.

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

Reproductive effects observed:

not specified

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Combined repeated/Screening reprotoxicity study complete and sufficient to fulfill the REACh annex VIII requirements.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 for the systemic toxicity. A reproduction and developmental NOAEL of at least 1000 mg/kg/day was derived.

Short description of key information:
In a study performed according to the OECD 422 (Notox 2011, 496701), 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 for the systemic toxicity. A reproduction and developmental NOAEL of at least 1000 mg/kg/day was derived.

Justification for selection of Effect on fertility via oral route:
Study performed according to the OECD guideline 422 and GLP compliant.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 July 2016 - 09 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name: N-(3-aminopropyl)iminodiethanol
Synonyms:
- Aminopropyldiethanolamine
- APDEA
- N-(3-Aminopropyl)diethanolamine
CAS No.: 4985-85-7
Batch No.: A290X2010101
Description: green liquid
Storage conditions: at room temperature and protected from humidity under nitrogen atmosphere
Purity: 80.83%
Expiry date: 01 September 2017

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, le Genest-Saint-Isle, France
- Age at study initiation: approximately 10-11 weeks old on the beginning of the treatment period
- Mean body weight at study initiation: a mean body weight of 305 g (range: 239 g to 358 g)
- Fasting period before study: no
- Housing: the animals were individually housed in polycarbonate cages (Tecniplast 2154, 940 cm2) with stainless steel lids and containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 4 or 5 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 16 January 2017 to 09 February 2017

Route of administration:

oral: gavage

Vehicle:

other: Drinking water treated by reverse osmosis

Details on exposure:

PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 16, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Type of method: High Performance Liquid Chromatography with tandem Mass Spectrometry detection (LC/MS-MS)
Test item concentrations: remained within an acceptable range of variations (+2.2% to +9.5%) when compared to the nominal values (± 10% of the nominal concentrations).
Stability: The dose formulations containing the test item and prepared as a solution at 2 mg/mL and 200 mg/mL in drinking water treated by reverse osmosis were found to be stable after 5 days of storage at room temperature.

Duration of treatment / exposure:

Day 6 to Day 20 p.c.

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

24 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for dose level selection:
The dose-levels were selected in agreement with the Sponsor, based on the results of a previous OECD 422 study. The test item was administered by daily oral gavage to male and female Wistar Han rats at dose-levels of 100, 300 and 1000 mg/kg/day.
At 1000 mg/kg/day, parental toxicity consisted of reduced locomotor activity (total movements) with a normal habituation pattern, reduced body weight gain for male animals, affected hematological parameters, changes in blood biochemistry parameters, and organ weight changes (increased liver, kidney and adrenal weights and decreased thymus weights) without accompanying microscopic findings. The only apparent treatment-related macroscopic and microscopic changes were in the stomach. This was probably related to irritancy of the test substance. There were several dark red or reddish foci on the glandular mucosa of the stomach at macroscopic examination, and microscopic stomach abnormalities consisting of acute inflammation, hyperplasia, degenerative vacuolation, necrosis and hemorrhage.
Up to 1000 mg/kg/day, no toxicologically relevant changes in body weight, body weight gain or food consumption were noted for the pregnant females. Mating, fertility and conception indices, pre-coital time, numbers of corpora lutea and implantation sites, gestation index, duration of gestation, and pup body weights were unaffected by treatment.
No treatment-related findings were observed at the low and mid dose-levels.
Based on these results and considering the microscopic changes in the stomach observed at 1000 mg/kg/day, 750 mg/kg/day was selected as the high dose-level. The low and intermediate dose levels were selected using a ratio representing an approximately 3-fold interval (i.e. 250 and 80 mg/kg/day).

- Rationale for animal assignment: computerized stratification procedure.

Maternal examinations:

MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period.

CLINICAL SIGNS:
- Time schedule: from arrival, each animal was observed once a day as part of the routine examinations. From the start of the treatment period, each animal was observed once a day, at approximately the same time.

BODY WEIGHT:
- Time schedule: the body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by each female was recorded for the following intervals: Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

POST-MORTEM MACROSCOPIC EXAMINATION:
- Sacrifice on Day 21 post-coitum.
- Examined: principal thoracic and abdominal organs

PRESERVATION OF TISSUES:
See Table 1
- For all study animals, samples of the stomach were collected and preserved in 10% buffered formalin.
- Samples of the macroscopic lesions observed in females (including those from the females euthanized prematurely) were preserved in 10% buffered formalin (or in another appropriate fixative). As remarkable macroscopic lesions were observed in test item-treated animals, samples of the corresponding tissues (kidneys, adipose tissue and adrenals) from five control animals were collected and preserved.

Ovaries and uterine content:

The ovaries and uterine content were examined after termination, including::
- gravid uterus weight,
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites,
- evaluation of placentas.

Fetal examinations:

- External examinations: Yes: all fetuses per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter
- Other : body weight, sex.

Statistics:

Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fisher’s exact probability test (proportions).

Indices:

% Pre-implantation loss = 100 * (Number of corpora lutea - Number of implantation sites) / Number of corpora lutea
% Post-implantation loss = 100 * (Number of implantation sites - Number of live fetuses) / Number of implantation sites

Historical control data:

Cf attached document

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See table 2.
Test item treatment-related clinical signs, also noted in prematurely sacrificed females, consisted of round back, piloerection and loud breathing in 1/22 surviving females given 750 mg/kg/day.

Ptyalism, reddish vaginal discharge, dacryhorrhea, area of hair loss on forelimb, nodosities, abscess, cutaneous lesions and/or scabs on the neck were considered to be unrelated to the test item treatment as they were reported on a limited number of occasions and/or without any dose-relationship.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were no test item treatment-related deaths in control animals or in the 80 and 250 mg/kg/day treated groups.

At 750 mg/kg/day, on Day 12 p.c., two pregnant females were prematurely sacrificed. Prior to death, these females showed signs of poor clinical condition (i.e. piloerection, emaciated appearance, abdominal and/or loud breathing, half-closed eyes and/or soft feces). Severe body weight loss was noted (between -43 to -64 g) over the last interval, together with reduced food consumption (1 to 8 g/animal/day).
At macroscopic post-mortem examination, the adrenal glands were enlarged (2/2 females) and brownish in color (1/2 females), the stomach mucosa showed several reddish discolored areas (2/2 females) and the cecum was dilated with gas (1/2 females). The right ureter, right kidney and right uterine horn were absent in one female.
These unscheduled deaths were considered to be test item treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See table 3.
At 750 mg/kg/day and when compared with controls, moderately lower mean body weight gain was noted on Days 9-12 p.c. (+11 g vs. +20 g in controls, p<0.05). This effect did not affect the mean body weight and did not correlate with any reduction in mean food intake.

At 80 or 250 mg/kg/day, there were no effects on mean body weight or mean body weight change.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

At 80, 250 or 750 mg/kg/day, there were no effects of treatment with the test item.

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, 1/22 females showed reddish colored foci on the forestomach. Since this finding was also observed in prematurely sacrificed females, it was considered to be test item treatment-related.

Other macroscopic findings (i.e. nodule in the right ovarian area, scabs and thickened subcutaneous tissue in the neck, unilateral absence of kidney and ureter together with unilateral kidney enlargement, unilateral atresia of uterine horn and/or enlarged placentae) were of isolated occurrence, not dose-related and/or due to spontaneous morphological malformations. They were, therefore, considered to be incidental.

Neuropathological findings:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

See table 5.
Net body weight change:
There were no dose-related effects on mean gravid uterus weight, carcass weight or net body weight change.

At 750 mg/kg/day and when compared with controls, females had a slightly lower mean net body weight change (+43 g vs. +50 g in controls); this was considered to be of no toxicological importance as this difference was minimal and did not correlate with any other finding.

Number of abortions:

no effects observed

Description (incidence and severity):

See table 4.

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.
There were no test item treatment-related effects.

At 750 mg/kg/day and when compared with controls, there was a slightly higher mean post-implantation loss (11.8% vs. 6.0%), along with a slightly higher mean number of early resorptions (1.2 vs. 0.8). This was mainly due to the contribution of two females, which had 80.0% and 64.3% post implantation losses, respectively. As these differences were not statistically significant, were close to the upper limit of the Historical Control Data and did not impact the mean number of live fetuses, a test item treatment relationship was considered to be unlikely.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

See table 6.

Early or late resorptions:

effects observed, non-treatment-related

Description (incidence and severity):

See table 6.
At 750 mg/kg/day and when compared with controls, there was a slightly higher mean post-implantation loss (11.8% vs. 6.0%), along with a slightly higher mean number of early resorptions (1.2 vs. 0.8). This was mainly due to the contribution of two females, which had 80.0% and 64.3% post implantation losses, respectively. As these differences were not statistically significant, were close to the upper limit of the Historical Control Data and did not impact the mean number of live fetuses, a test item treatment relationship was considered to be unlikely.

Dead fetuses:

no effects observed

Description (incidence and severity):

See table 6.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

See table 4.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

clinical signs

mortality

Abnormalities:

effects observed, non-treatment-related

Fetal body weight changes:

no effects observed

Description (incidence and severity):

See table 7.
There were no effects on mean fetal body weight at any dose level.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

See table 7.
There were no effects on sex-ratio (percentage of male fetuses) at any dose level.

External malformations:

effects observed, treatment-related

Description (incidence and severity):

See table 8.
Variations
There were no external variations at external fetal examination.

Malformations
In the 750 mg/kg/day group, two fetuses from two different litters had external malformations:
- one fetus (fetal body weight: 4.42 g): anasarca, cleft palate, mandibular micrognathia, short digits, micromelia, short trunk [split supraoccipital, split palate and absent hindpaw phalanx (at skeletal examination], bent tail and gastroschisis,
- one fetus (fetal body weight: 5.30 g): omphalocele.

These findings, mainly affecting the trunk (mid-line defects: gastroschisis or omphalocele), were observed in two malformed fetuses from two different litters, and one of these fetuses also had a body weight significantly lower than the other fetuses. A test item treatment-related effect cannot be ruled out taking into account a potential dose-relationship although the incidences of these malformations were similar to the Historical Control Data.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

See tables 11 to 12.
Cartilage
There were no test item treatment-related cartilage observations.
Cartilage findings [i.e. cartilage of cervical vertebra(e) present and cartilage of distal phalanx present] were observed with a higher litter incidence in the 250 mg/kg/day group and were considered to be unrelated to the test item treatment as they were not dose-related.

Variations
At 250 and 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of the interparietal bone for which the differences from controls were statistically significant at 750 mg/kg/day.

At 750 mg/kg/day and when compared with controls, there was a statistically significant increase in delayed ossification of the supraoccipital and parietal bones (incomplete ossification).

These variations, mainly affecting the head bones from 250 mg/kg/day, were considered to be associated with the test item treatment, although the incidences were below the maximum values in the Historical Control Data.

Other skeletal variations, (i.e. unossified distal phalanx of the forepaw was observed with a statistically significant higher litter incidence in the 250 mg/kg/day group) were considered to be unrelated to the test item treatment as they were not dose-related.

Malformations
In the 750 mg/kg/day group, one litter had a malformed fetus, with split palate, split supraoccipital and absent hindpaw phalanx (along with cleft palate, mandibular micrognathia, short digits and micromelia at external examination).

In the 250 mg/kg/day group, one litter had a malformed fetus (fetal body weight 2.97 g), with bent ulna and bent radius (along with misshapen cartilage of the radius and ulna).

In the 80 mg/kg/day group, no malformed fetuses were observed.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

See tables 9 and 10.
Variations
Soft tissue variations (i.e. dilated renal pelvis, short or absent innominate artery, malpositioned artery, dilated ureter, and/or reddish foci on the thymus) were not dose-related and/or were observed with a similar or minimally different incidence in control and/or test item treated groups. Therefore, they were considered to be unrelated to the test item treatment.

Malformations
There was no test item treatment-related increase in the frequency of soft tissue malformations.
In the 250 mg/kg/day group, one litter contained one malformed fetus with no left kidney and left ureter. As this isolated malformation was not dose-related and was within the Historical Control Data, it was considered to be of no toxicological importance.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

external malformations

skeletal malformations

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

external: face

external: limb

external: paw

external: tail

external: trunk

external: thorax

external: umbilicus

skeletal: skull sutures

skeletal: hindlimb

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

 

TISSUE PROCEDURE TABLE

Table 1: Preservation of tissues 

Organs	Organ weights	Preservation of tissues	Microscopic examination
Macroscopic lesions		X
Stomach		X

 

Table 2: Clinical signs

 

Dose-level (mg/kg/day)	0	80	250	750
Round back				1 (on Day 15p.c.)
Piloerection				1 (on Day 15p.c.)
Loud breathing				1 (from Day 13 to 19p.c. )
Number of affected animals	0/24	0/24	0/24	1/22

( ): in brackets: Days p.c. of occurrences.

Table 3: Body weight

 

Dose-level (mg/kg/day)	0	80	250	750
Body weight (g)
Day 6 p.c.	302	305	305	306
	-	(+1)	(+1)	(+1)
Day 9 p.c.	318	322	322	323
	-	(+1)	(+1)	(+2)
Day 12 p.c.	338	344	344	334
	-	(+2)	(+2)	(-1)
Day 15 p.c.	357	363	365	360
	-	(+2)	(+2)	(+1)
Day 18 p.c.	398	404	409	397
	-	(+2)	(+3)	(0)
Day 21 p.c.	448	453	463	447
	-	(+1)	(+3)	(0)
Body weight change (g)
Days 6 - 9 p.c.	+16	+17	+17	+17
Days 9 - 12 p.c.	+20	+22	+22	+11*
Days 12 - 15 p.c.	+18	+18	+21	+19
Days 15 - 18 p.c.	+42	+41	+44	+38
Days 18 - 21 p.c.	+50	+49	+53	+50
Days 6 - 21 p.c.	+146	+148	+158	+140
	-	(+1)	(+8)	(-4)

Statistical significance;^*: p<0.05.

p.c.        :post-coitum.

-             : not applicable.

( )           : in brackets, percentage differencevs.controls.

Table 4: Pregnancy status

 

Dose-level(mg/kg/day)	0	80	250	750
Number of females	24	24	24	24
Non-pregnant females	0	0	1	0
Prematurely sacrificed females	0	0	0	2
Females with total resorption	0	0	0	0
Females with live fetuses at term	24	24	23	22

 

Table 5: Mean carcass, net change and gravid uterus weights (a) (g)

 

Dose-level (mg/kg/day)	0	80	250	750
Gravid uterus weight	96 -	95 (-1)	105 (+9)	98 (+2)
Carcass weight	352 -	358 (+2)	357 (+1)	350 (-1)
Net body weight change from Day 6.p.c.	+50	+53	+53	+43

( ): in brackets, percentage differencevs.controls.

p.c.        :post-coitum.

(a): weights are rounded values.

-             : not applicable.

Table 6: Hysterectomy data

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
Number of pregnant females at hysterectomy	24	24	23	22	407
Number of females with live fetuses at termination	24	24	23	22	388
Number of females with total resorption	0	0	0	0	0
Mean number ofcorpora lutea	14.2	14.9	16.3	15.0	[13.8; 16.0]
Mean number of implantation sites	13.0	12.6	14.4	13.3	[12.5; 14.5]
Mean pre-implantation loss (%)	10.0	14.9	11.4	10.5	[6.2; 14.0]
Mean number of live fetuses	12.2	11.7	13.3	12.0	[11.6; 13.8]
Dead fetuses (mean %)	0.0	0.0	0.0	0.0	[0.00; 0.50]
Mean number of implantation scars	0.0	0.0	0.0	0.0	/
Mean number of early resorptions	0.8	0.9	1.0	1.2	/
Mean number of late resorptions	0.1	0.0	0.2	0.1	/
Mean post-implantation loss (%)	6.0	7.7	8.6	11.8	[3.5; 11.1]

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

/              : not reported in HCD.

Table 7: Fetal examinations ( Body weight and sex)

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
Mean fetal body weight (g)	5.80 -	5.88 (+1)	5.72 (-1)	5.91(+2)	[5.5; 5.9]
Mean fetal body weight of males (g)	5.87 -	6.06 (+3)	5.88 (0)	6.07(+3)	[5.7; 6.1]
Mean fetal body weight of females (g)	5.70 -	5.69 (0)	5.57 (-2)	5.74(+1)	[5.4; 5.7]
Mean percentage of male fetuses (%)	47.4	51.8	47.5	50.0	[44.0; 55.4]

( ): in brackets, percentage differencevs.controls.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016), [min.; max.].

-             : not applicable.

Table 8: Fetal (litter) incidences (%) of external malformations

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
Dams with live fetuses	24	24	23	22	388
Number of live fetuses	292	280	305	264	5000
Litters affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	2 (9.1)	11 (2.8)^(b)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	2 (0.8)	13 (0.3)^(b)
Anasarca, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	/
Cleft palate, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)
Mandibular micrognathia, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)
Short digit(s), F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)
Micromelia, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)
Gastroschisis, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.6)^(a)
Short trunk, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)
Omphalocele, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.3)^(a)
Bent tail, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.4 (4.5)	0.4 (5.0)^(a)

F            : fetal incidence.

L            : litter incidence.

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in HCD.

^(a)           : maximum incidence.

^(b)           : mean incidence.

Table 9: Fetal (litter) incidences (%) of soft tissue variations

 

Dose-level (mg/kg/day)		0	80	250	750	HCD
Dams with live fetuses	24		24	23	21	387
Number of live fetuses	139		136	145	125	2404
Litters affected, n (%)	10 (41.7)		8 (33.3)	6 (26.1)	7 (33.3)	86 (22.2) (b)
Fetuses affected, n (%)	23 (16.5)		9 (6.6)	12 (8.3)	14 (11.2)	119 (5.0) (b)
Dilated renal pelvis, F (L)	2.9 (12.5)		1.5 (8.3)	5.5 (21.7)	4.0 (9.5)	9.5 (28.6) (a)
Short innominate artery, F (L)	1.4 (8.3)		0.7 (4.2)	0.7 (4.3)	1.6 (9.5)	3.7 (22.7) (a)
Absent innominate artery, F (L)	2.9 (16.7)		0.7 (4.2)	0.0 (0.0)	2.4 (14.3)	5.1 (25.0) (a)
Malpositioned subclavian artery, F (L)	0.0 (0.0)		0.0 (0.0)	0.0 (0.0)	0.8 (4.8)	/
Dilated ureter, F (L)	10.8 (20.8)		3.7 (20.8)	4.8 (8.7)	4.8 (14.3)	7.1 (28.0) (a)
Thymus: reddish foci, F (L)	0.0 (0.0)		0.7 (4.2)	0.0 (0.0)	0.0 (0.0)	/

F: fetal incidence.

L: litter incidence.

HCD    : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/: not reported in Historical Control Data.

(a): maximum incidence.

(b): mean incidence.

Table 10: Fetal (litter) incidences (%) of soft tissue malformations

 

Dose-level (mg/kg/day)	0	80	250	750		HCD
Dams with live fetuses	24	24	23	21		387
Number of live fetuses	139	136	145	125		2404
Litters affected, n (%)	0 (0.0)	0 (0.0)	1 (4.3)	0 (0.0)	7 (1.8)^(b)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	1 (0.7)	0 (0.0)	13 (0.5)^(b)
Absent kidney, F (L)	0.0 (0.0)	0.0 (0.0)	0.7 (4.3)	0.0 (0.0)	0.7 (4.5)^(a)
Absent ureter, F (L)	0.0 (0.0)	0.0 (0.0)	0.7 (4.3)	0.0 (0.0)	/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in Historical Control Data.

^(a)           : maximum incidence.

^(b)           : mean incidence.

Table 11: Fetal (litter) incidences (%) of skeletal variations

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
Dams with live fetuses	24	24	23	22	388
Number of live fetuses	153	144	160	139	2596
Supraoccipital: incomplete ossification, F (L)	0.0 (0.0)	0.7 (4.2)	0.0 (0.0)	3.6* (9.1)	5.6 (25.0)^(a)
Parietal: incomplete ossification, F(L)	0.7 (4.2)	0.0 (0.0)	0.6 (4.3)	4.3*(22.7)	9.2 (35.0)^(a)
Interparietal: incomplete ossification, F(L)	0.0 (0.0)	0.7 (4.2)	1.9 (13.0)	5.0** (18.2*)	9.2 (45.0)^(a)

F: fetal incidence.

L: litter incidence.

Statistically significant; *: p<0.05 and **: p<0.01 for the number of fetuses or litters affected or for affected fetuses/litter (incomplete ossification of parietal).

HCD      : Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a)           : maximum incidence.

Table 12: Fetal (litter) incidences (%) of fetal skeletal malformations

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
Dams with live fetuses	24	24	23	22	388
Number of live fetuses	153	144	160	139	2596
Litters affected, n (%)	0 (0.0)	0 (0.0)	1 (4.3)	1 (4.5)	15 (3.9)^(b)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	1 (0.6)	1 (0.7)	18 (0.7)^(b)
Supraoccipital: split, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.7 (4.5)	/
Palate: split, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.7 (4.5)	0.7 (5.0)^(a)
Ulna: bent, F(L)	0.0 (0.0)	0.0 (0.0)	0.6 (4.3)	0.0 (0.0)	/
Radius: bent, F(L)	0.0 (0.0)	0.0 (0.0)	0.6 (4.3)	0.0 (0.0)	/
Hindpaw: absent phalanx, F (L)	0.0 (0.0)	0.0 (0.0)	0.0 (0.0)	0.7 (4.5)	/

F: fetal incidence.

L: litter incidence.

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

/              : not reported in Historical Control Data.

^(a)           : maximum incidence.

^(b)           : mean incidence.

Table 13:

Distribution of fetal malformations

 

Dose-level (mg/kg/day)	0	80	250	750	HCD
External
Litters affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	2 (9.1)	11 (2.8)^(a)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	0 (0.0)	2 (0.8)	13 (0.3)^(a)
Soft tissue
Litters affected, n (%)	0 (0.0)	0 (0.0)	1 (4.3)	0 (0.0)	7 (1.8)^(a)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	1 (0.7)	0 (0.0)	13 (0.5)^(a)
Skeletal
Litters affected, n (%)	0 (0.0)	0 (0.0)	1 (4.3)	1 (4.5)	15 (3.9)^(a)
Fetuses affected, n (%)	0 (0.0)	0 (0.0)	1 (0.6)	1 (0.7)	18 (0.7)^(a)
Total
Litters affected / evaluated (%)	0/24 (0.0)	0/24 (0.0)	2/23 (8.7)	2/22 (9.1)	/
Fetuses affected / evaluated (%)	0/292 (0.0)	0/280 (0.0)	2/305 (0.7)	2/264 (0.8)	/

HCD: Historical Control Data (control data collected from 18 studies covering a period ranging from March 2014 to July 2016).

^(a): mean incidence.

/: not reported in HCD.

Conclusions:

The test item was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to pregnant Sprague-Dawley rats at 80, 250 and 750 mg/kg/day. The control group received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions.

On the basis of the results obtained in this study:
- the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day (based on poor clinical condition leading to premature euthanasia at 750 mg/kg/day),
- the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day (based on 2 fetuses with fetal malformations at 750 mg/kg/day) in a context of severe maternal toxicity.

Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item on the pregnant female and on embryonic and fetal development, following daily oral administration (gavage) to pregnant female rats from implantation until the day before scheduled hysterectomy [Day 6 to Day 20 post-coitum (p.c.) inclusive].

This GLP study was carried out according to OECD test guideline No. 414 (22 January 2001).

 

Methods

Three groups of 24 time-mated female Sprague-Dawley rats received the test item by the oral route (gavage), at 80, 250 or 750 mg/kg/day, once daily from Day 6 to Day 20 p.c. inclusive. Another group of 24 time-mated rats received the vehicle only, drinking water treated by reverse osmosis, under the same experimental conditions, and acted as a control group. A constant dosage volume of 5 mL/kg/day was used. The test item concentrations were determined in the dose formulations.The animals were checked daily for mortality and clinical signs. Body weight and food consumption were recorded over designated intervals. On Day 21 p.c., the females were sacrificed and a macroscopic post-mortem examination was performed. Hysterectomy was performed and the numbers of corpora lutea, implantation sites, uterine scars, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities (including cartilage).

Samples of stomach and macroscopic lesions were collected from all dams and preserved in 10% buffered formalin.

 

Results

Chemical analyses

The concentrations of the test item in the dose formulations remained within acceptable ranges of variations (+2.2% to +9.5%) when compared with the nominal concentrations (± 10%).

The test item concentration was below the Limit Of Quantification (0.01 mg /mL) in control dose formulations.

 

Pregnancy status

There were 24, 24, 23 and 22 females with live fetuses in the groups given 0, 80, 250 and 750 mg/kg/day, respectively.

 

Mortality

At 750 mg/kg/day, two pregnant females were prematurely sacrificed due to signs of poor clinical condition (piloerection, emaciated appearance, abdominal and/or loud breathing, half-closed eyes and/or soft feces, and severe body weight loss together with reduce food intake).At necropsy, the adrenal glands were enlarged and/or brownish in color, the stomach mucosa showed several reddish discolored areas and/or the cecum was dilated with gas.These findings were considered to be test item treatment-related.

No other death occurred in the low and mid dose groups.

 

Clinical signs

Test item treatment-related clinical signs were transient and consisted of round back, piloerection and loud breathing in 1/22 females given 750 mg/kg/day.

Body weight

There was no effect on mean body weight at any dose-level.

At the high dose-level of 750 mg/kg/day, mean body weight gain was significantly lower than controls

(-45%) on Days 9-12 p.c. (+11 g vs. +20 g, p<0.05).

 

Food consumption

There were no test item treatment-related effects at any dose-level.

 

Necropsy and macroscopic post-mortem examination

Reddish colored foci on the forestomach mucosa was observed in 1/22 females given 750 mg/kg/day and sacrificed as scheduled. No test item-related findings were observed in the low and mid dose-level groups.

 

Net body weight change

There were no dose-related effects on mean gravid uterus weight, carcass weight or net body weight change at any dose-level.

 

Hysterectomy data

There were no test item treatment-related effects.

Fetal body weight and percentage of male fetuses

There were no test item treatment-related effects.

 

Fetal examinations

External examination

There were no test item-related external or oral cavity variations at examination of the fetuses at any dose-level. At 750 mg/kg/day, two fetuses, from two different litters had anasarca, cleft palate, mandibular micrognathia, short digits, micromelia, short trunk, bent tail, gastroschisis and/or omphalocele for which a relationship to the test item could not be ruled out.

 

Soft tissue examination

There were no test item-related variations or malformations at soft tissue examinationat any dose-level.

 

Cartilage and skeletal examinations

At 250 and 750 mg/kg/day and when compared with controls, there were higher litter and fetal incidences of fetuses with delayed ossification: incomplete ossification of the interparietal bone (statistically significant at 750 mg/kg/day).

 

At 750 mg/kg/day and when compared with controls, there were statistically significant increases in litter and fetal incidences of fetuses with skeletal abnormalities mainly involving the head bones, such as incomplete ossification of the supraoccipital and parietal bones (variations), split palate and split supraoccipital (malformations observed in one fetus also showed an absence of the hindpaw phalanx).

Conclusion

The test item was administered by gavage, once daily, from Days 6 to 20 p.c., inclusive, to pregnant Sprague-Dawley rats at 80, 250 and 750 mg/kg/day. The control group received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions.

 

On the basis of the results obtained in this study:

.         the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 250 mg/kg/day (based on poor clinical condition leading to premature euthanasia at 750 mg/kg/day),

.         the NOAEL for embryo-fetal development was considered to be 250 mg/kg/day (based on 2 fetuses with fetal malformations comparable to HCD at 750 mg/kg/day) in a context of maternal toxicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

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. Therefore, no classification is required according to CLP and DSD criteria.

In a study performed accroding to OECD 414, pregnant rats were administered the test item during says 6 to day 19 p.c.

In terms of maternal toxicity, the following test item treatment-related findings were recorded at 750 mg/kg/day:

.         mortality: two prematurely sacrificed animals (on Day 12p.c.) showed severe clinical signs, body weight loss and reduced food consumption and necropsy findings prior to sacrifice,

.         clinical signs: round back, piloerection and loud breathing (also noted in prematurely sacrificed females) in 1/22 surviving females in the second week of treatment.

In terms of embryo/fetal development, the following test item treatment-related findings were recorded:

 

At 750 mg/kg/day:

.         fetal examination: when compared with controls and/or Historical Control Data, increases in the incidences of external malformations observed in two fetuses from two different litters (for which most of the malformation incidences were similar to the Historical Control Data): anasarca, cleft palate, mandibular micrognathia, short digits, micromelia, short trunk, bent tail, gastroschisis and/or omphalocele, and increases in the incidences of skeletal findings, mainly involving the head bones (variations: incomplete ossification of the interparietal, parietal and supraoccipital bones for which incidences were below the maximum values in the Historical Control Data; skeletal malformations: split palate, split supraoccipital and absent hindpaw phalanx).

 

At 250 mg/kg/day:

.         fetal examination: when compared with controls and/or Historical Control Data, increases in the incidence of skeletal abnormalities mainly impacting the head bones (variations: incomplete ossification of the interparietal bone for which the incidence was below the maximum values in the Historical Control Data).

 

Overall, at 750 mg/kg/day, in a context of severe maternal toxicity (poor clinical condition leading to premature euthanasia), there was a series of fetal findings that mainly impacted the abdomen and the head bones of two fetuses (one in each litter). A test item treatment-related effect was considered to be unlikely for malformations observed at 250 mg/kg/day (i.e.ureter and kidney agenesis, bent ulna and/or bent radius).

On the basis of the results obtained in this study,  the No Observed Adverse Effect Level (NOAEL) for maternal parameters and for embryo fetal development was considered to be 250 mg/kg/day.

Based on these results no classification according to CLP and GHS is warranted for developmental toxicology