N-(3-aminopropyl)iminodiethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

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: APDEA (N-(3-Aminopropyl)diethanolamine)
-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: 309-311 g for males, 199-203 for females
- Fasting period before study: no
- 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.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Elix, Millipore S.A.S., Molsheim, France

Details on oral 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.

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:

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.

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.

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 (Main) and 5 males and 5 females for Recovery (control and high dose only).

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

Observations and examinations performed and frequency:

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.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean diet consumption calculated as g food/kg body weight/day: Yes, weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: from Main and recovery group, prior to scheduled post mortem examination, and from all Recovery animals at the end of treatment.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, overnight
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: from Main and recovery group, prior to scheduled post mortem examination, and from all Recovery animals at the end of treatment.
- Animals fasted: Yes / No / No data
- How many animals: all
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at pre-test and during Week 4 of treatment.
- Dose groups that were examined: all Main and Recovery animals. Based on possible treatment-related findings, all Recovery animals were also tested at the end of the recovery phase.
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, grip strength, locomotor activity

Sacrifice and pathology:

SARIFICE
-Main animals: After at least 28 days of dose administration.
-Recovery animals: After the recovery period of at least 14 days, which was at least 14 days after the scheduled kill of the Main animals.
ORGAN WEIGHT: Yes (see list 4)
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (see list 3)

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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period.
Salivation seen after dosing for most animals at 1000 mg/kg during most of the treatment period and one female rat at 300 mg/kg during the first week of treatment was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance.
Alopecia was noted for single animals, which was within the range of background for rats of this age and strain which are housed and treated under the conditions in this study. In addition, one female rat (number 96) showed exophthalmos and opacity of the right eye. This was considered due to the method of blood sampling and not considered treatment related.

BODY WEIGHT AND WEIGHT GAIN
Body weights and body weight gain were slightly reduced for males treated at 1000 mg/kg throughout the treatment period. Statistical significant differences were noted for body weights on Day 29 of study, and for body weight gain on Days 8-29 of study. The reduction in body weight gain compared to controls was approximately 25-30%. Complete recovery was noted during the two-weeks treatment free period.
No toxicologically relevant changes in body weights and body weight gain were noted for males at 100 and 300 mg/kg and for females treated up to 1000 mg/kg.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
At the end of treatment, several haematological parameters were affected at 1000 mg/kg. For both sexes, decreased values for monocytes, red blood cells, haemoglobin, haematocrit, and mean corpuscular volume (MCV), and increased prothrombin time (PT) were noted. In addition at this dose, male rats showed increased concentration of white blood cells, and female rats showed decreased percentage of neutrophils and increased percentage of lymphocytes. All values recovered to normal within the two-week treatment free period.
At the end of recovery, increased concentration of platelets was noted for males at 1000 mg/kg when compared to the control group. However, as this finding was not seen at the end of treatment and the values were within normal limits, it was not considered toxicologically relevant.
All other statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

CLINICAL CHEMISTRY
At the end of treatment, several clinical biochemistry parameters were affected at 1000 mg/kg. For both sexes, increased values for aspartate aminotransferase (ASAT), urea and bile acids were noted. Male rats treated at 1000 mg/kg showed increased values for alanine aminotransferase (ALAT) and total bilirubin, and decreased concentration of total protein and albumin. In addition, increased potassium levels were noted for female rats at the high dose.
Furthermore, calcium concentration was slightly increased for females at 300 and 1000 mg/kg, and was just outside the historical control data.
All affected parameters showed recovery after two weeks without treatment.
At the end of recovery, decreased potassium concentration was observed for males, and decreased creatinine level and increased sodium concentration were seen for females at 1000 mg/kg when compared to the control group. However, as these findings were not seen at the end of treatment and
the values were within normal limits, it was not considered toxicologically relevant.
The other statistically significant changes noted for females at the end of treatment (total protein at 300 mg/kg and cholesterol at all dose groups) were considered to be of no toxicological relevance as they occurred in the absence of a dose response relationship, were considered to have arisen as a result of slightly high control values and/or remained within the range considered normal for rats of this age and strain.

NEUROBEHAVIOUR
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
For the motor activity test at Week 4 of study, reduced total movements were noted for males and females at 1000 mg/kg.
Mean number of ambulations were reduced for both sexes at 1000 mg/kg and for females at 300 mg/kg. However, the number of ambulations for all occasions were within normal limits and for the females the statistical significant finding was considered due to the high value of one control female
(number 64). Therefore, it is considered not toxicologically significant.
After two weeks of recovery, total movements and ambulations were comparable for control and high dose animals.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.
The statistical significant findings noted at pretest were considered to have occurred by chance, as treatment was not started yet and all data was within normal limits.

ORGAN WEIGHTS
At 1000 mg/kg, several organ weight changes were noted. These consisted of increased kidneys weights (absolute and relative) for both sexes at the end of treatment and recovery, increased liver weights (absolute and/or relative) for both sexes at the end of treatment, and also for Main females at the end of recovery, increased adrenals weight (relative for males and absolute for Repro females) at the end of treatment, and decreased relative thymus weights for Repro females.
In addition, increased absolute and/or relative kidneys weights were also 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. Terminal body weight was decreased for males of the high dose group at the end of treatment, which was consistent with the in-life results. This lower terminal body weight caused an increase in relative brain weight, and was therefore not considered toxicological relevant.
The increased terminal body weight observed for Repro females at the same dose group was not considered toxicological significant as this slight increase in body weight was not regarded adverse.
All other statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution, was only noted at the end of recovery (not at the end of treatment), and/or remained within the range considered normal for rats of this age and strain.

GROSS PATHOLOGY
There was no toxicologically relevant findings indicating a sign of systemic toxicity. The only effect noted in the stomach at 1000 mg/kg was considered to be related to irritancy of the test substance. At 1000 mg/kg, macroscopic observation at necropsy revealed stomach abnormalities (several dark red or reddish foci at the glandular mucosa) for three males at the end of treatment. At histopathological examination this finding was confirmed as haemorrhage.
For a few animals of Groups 1, 2 and 3, also reddish foci were noted at the glandular mucosa of the stomach. However, as these were isolated foci and no toxicological relevant findings were noted at microscopic examination, these findings were not considered toxicological relevant.
Other findings that were noted among control and/or treated animals were considered to be of no toxicological significance, since they remained within the range of biological variation for rats of this age and strain. In addition, watery fluid in the uterus, found in ten females distributed over all groups, is related to a stage in the oestrous cycle and is a normal finding.

HISTOPATHOLOGY: NON-NEOPLASTIC
No toxicologically relevant microscopic findings that indicated a sign of systemic toxicity were noted in any treatment groups. The only apparent treatment related findings in unmated main study animals were limited to stomach findings which are probably due to irritant property of test substance. Minimal to moderate acute inflammation (7 males and 5 females), minimal or slight hyperplasia of nonglandular epithelium (7 males and 6 females), and minimal or slight increased vacuolative degeneration of the non-glandular stomach (2 males and 5 females) were noted at 1000 mg/kg. One male had a slight necrosis and minimal or slight haemorrhage was noted in 3 males and one female.
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 at the control and 100 mg/kg group. Several stomach observations occurred in the region of the limiting ridge (margo-plicatus) where the mucosal surface transitions from squamous to glandular. It should be noted that thickening (hyperplasia) of the nonglandular 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. Inflammation in the stomachs was limited to 1000 mg/kg animals and was primarily associated with the glandular mucosa. Following a 14 day recovery period, most findings were subsidized and no necrosis or haemorrhage was noted in previously treated animals. Minimal or slight acute inflammation was present in 2 males and one female at 1000 mg/kg.
While hyperplasia of the non-glandular epithelium was comparable between control and 1000 mg/kg males, these was a slight increased incidence in 1000 mg/kg females.
Staging of spermatogenesis in testes did not provide any evidence of test article related impairment to the spermatogenetic cycle. There was no evidence of reproductive toxicity.
Most microscopic findings recorded were considered to be within the normal range of background pathology encountered in Wistar (Han) rats of this age and strain.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: treatment related macroscopic and microscopic changes in the stomach related to irritancy of the test substance. dark red or reddish foci at the glandular mucosa of the stomach at macroscopic examination, and microscopic stomach abnormalities.

2

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

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.

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. 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).

Based on these results, a parental No Observed Adverse Effect Level (NOAEL) of 300 mg/kg was established.