N-(3-aminopropyl)iminodiethanol

Endpoint summary

Description of key 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.

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

In a study performed according to OECD 408, the test item was administred by oral route during 13 weeks followed by a 6 -week treatement free period. Under the experimental conditions of the study, the NOAEL (No Observed Adverse Effect Level) was established at 250 mg/kg/day based on multiple correlated effects observed at 650 mg/kg/day indicative of an impact on red blood cell mass and adverse microscopic lesions in several organs (erosions/ulcers in the forestomach and/or stomach in both sexes and tubular degeneration/necrosis in kidneys).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

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

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.

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.

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

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.

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.

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.

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'

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

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.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 June 2016 - 06 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

21 September 1998

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:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: breeder: Charles River Laboratories France, France
- Age at study initiation: approximately 5 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 171 g (range: 135 g to 198 g) and the females had a mean body weight of 121 g (range: 104 g to 141 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes (same sex and group), in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) 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 11 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: 26 September 2016 to 06 February 2017.

Route of administration:

oral: gavage

Vehicle:

other: Drinking water treated by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 5, 25 and 65 mg/mL
- Amount of vehicle (if gavage): 10 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 -3.4% 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:

13 weeks of treatment (followed by a 6-week treatment-free period)

Frequency of treatment:

Daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

15 animals per sex (for control and high dose groups)
10 animals per sex (for low and intermediate dose groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
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 bw/day followed by a 14-day recovery period. At 1000 mg/kg bw/day, parental toxicity consisted of reduced locomotor activity (total movements) with a normal habituation pattern, reduced body weight gain for males, affected hematological and clinical biochemistry parameters, and organ weight changes (increased liver, kidney and adrenal weights and decreased thymus weights). The only apparent test item treatment-related macroscopic and microscopic changes were in the stomach and these were probably related to test substance irritancy. No toxicologically significant changes were noted at 300 or 100 mg/kg bw/day.

- Rationale for animal assignment: computerized randomization procedure.

Positive control:

no (not required)

Observations and examinations performed and frequency:

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

CLINICAL OBSERVATIONS:
- Time schedule: each animal was observed once a day, at approximately the same time.

DETAILED CLINICAL EXAMINATION:
- Time schedule: detailed clinical examinations were performed on all animals, once before the beginning of the treatment period and then once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: all main animals were evaluated once in Week 12 (before the daily treatment).

Functional Observation Battery (FOB)
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and the measurement of motor activity.
The animals were randomized and all animals were observed in the cage, in the hand and in the standard arena.

Motor activity:
For each animal, motor activity was measured by automated infra-red sensor equipment over a 60 minute period.

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, then on the first day of treatment and at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week, over a 7 day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals, before the beginning of the treatment period and on all main control and high-dose animals on one occasion at the end of the treatment period.

HAEMATOLOGY:
- Time schedule for peripheral blood: the parameters were determined for all animals sacrificed at the end of the treatment period or treatment-free period and the animal found dead after blood sampling.
- Time schedule for bone marrow: two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all animals sacrificed at the end of the treatment period or treatment-free period and animal found dead after blood sampling.
- Parameters checked in table [No. 2] were examined.
- Animals fasted: Yes
- How many animals: all

THYROID HORMONES:
- Time schedule: an additional blood sample was taken from each animal sacrificed at the end of the treatment or treatment-free period and from the animal found dead after blood sampling.
The levels of the thyroid hormones T3 and T4 (determination by LC MS/MS) and thyroid stimulating hormone TSH (determination by ELISA) were not determined as there was no indication of an effect on the pituitary-thyroid axis.
- Animals fasted: Yes

URINALYSIS:
- Time schedule for collection of urine: the parameters were determined for all animals sacrificed at the end of the treatment period or treatment-free period and animal found dead after blood sampling.
- Parameters checked in table [No. 3] were examined.

Sacrifice and pathology:

ORGAN WEIGHTS: see table 4.
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment-free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

PRESERVATION OF TISSUES:
For all study animals, the tissues specified in the Tissue Procedures Table were preserved in 10% buffered formalin (except for the eyes and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).
Two bone marrow smears for potential determination of the bone marrow differential cell count were prepared from the femur of each animal euthanized on completion of the treatment or treatment-free period.

PREPARATION OF HISTOLOGICAL SLIDES
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).

HISTOPATHOLOGY:
A microscopic examination was performed on all tissues listed in the Tissue Procedure Table:
- for the control and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period and for the animal found dead,
- for all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.

According to the microscopic results of the high-dose animals, the following tissues were examined:
- liver, kidneys, stomach (with forestomach) and spleen from low- and intermediate-dose males and females (groups 2 and 3) euthanized at the end of the treatment period and from recovery males and females (groups 1 and 4),
- adrenals from low- and intermediate-dose males (groups 2 and 3) euthanized at the end of the treatment period and from recovery males (groups 1 and 4),
- histological slides stained according to PAS procedures (liver and kidneys in control and high dose animals at the end of the treatment period).

Statistics:

Citox software was used to perform the statistical analysis of body weight, food consumption, motor activity, hematology, blood biochemistry and urinalysis data.
PathData software was used to perform the statistical analysis of organ weight data.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See Table 5.
At 650 mg/kg/day, 2/15 females had thin appearance for 10 or 13 days, together with piloerection and hunched posture for 25 days in one of these females.
Ptyalism was also transiently observed in 3/15 males and 2/15 females.
These test item-related clinical signs were considered to be of minor toxicological importance as they were of isolated occurrence, reported only sporadically and/or observed in 1/15 control females from Day 124 (Week 18) (hunched posture and thin appearance).

Reflux at administration was noted in 4/10 females given 250 mg/kg/day and 1/15 males and 3/15 females given 650 mg/kg/day on one to three occasions. Although reflux at administration was also noted in 2/15 control males and 1/15 control females, these findings were considered to be test item-related and most probably linked to the alkaline values of dose formulations.

The test item treatment-related clinical signs were reversible as they were no longer observed over the treatment-free period.

The other clinical signs recorded during the study, i.e. alopecia, scabs, thinning of hair, hypoactivity, dyspnea, chromorhynorrhea and/or, hunched posture were of isolated occurrence, observed both in control and test item-treated animals, and/or showed no dose-relationship.
These clinical signs were therefore considered to be unrelated to the test item treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test item treatment-related deaths occurred during the study.

At 50 mg/kg/day, one female was found dead after blood sampling for laboratory investigations on Day 89 (Week 13). No findings were observed at necropsy or at any examination, therefore the death was not considered as treatment-related but most probably due to the blood sampling procedures.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See Table 6.
At 650 mg/kg/day, in males and females, the mean body weight gain recorded during the Days 1-91 interval was statistically significantly lower than that of the controls (-11% and -18%, respectively). This treatment-related effect led to a minimally lower mean body weight in both sexes towards the end of the treatment period (-7% and -8% vs. controls in males on Days 78 and 85, respectively, and -7% on Day 91 in females, p<0.05). The differences from controls were particularly noticeable from the second month in females and in the third month in males. This effect was attributed to the test item treatment. During the recovery period, these changes were progressive reversible in both sexes.

At 250 mg/kg/day in females, when compared with controls, the slightly lower mean body weight gain was not considered to be toxicological significant as it was of minor amplitude and not statistically significant.
No relevant effects were noted on body weight or body weight gain in males given 250 mg/kg/day.

At 50 mg/kg/day, no relevant effects were noted on body weight or body weight gain in males or females during the study period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See Table 7.
At 650 mg/kg/day, in males and females, when compared with controls, statistically significant, lower mean food consumption was recorded on some occasions during the treatment period (i.e. in Weeks 5, 6, 9 and/or 12). This mainly correlated with the minimally lower terminal mean body weight recorded in males. This change was reversible at the end of the treatment-free period.

At 50 and 250 mg/kg/day, no relevant effects were observed on food consumption in males or females during the study period.

Food efficiency:

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item treatment-related findings were observed at the end of the treatment period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 8.
End of treatment period
At 650 mg/kg/day
When compared with controls, a moderate decrease in red blood cell mass was observed in males and females. These findings were considered to be adverse in view of their magnitude and their correlation with histopathological findings (decreased hematopoiesis, mainly erythropoieisis, in the spleen and to a lesser extent in the liver).
This decrease in red blood cell mass in both sexes was characterized by decreases in red blood cell count, mean hemoglobin concentration, mean packed cell volume, mean cell volume and mean cell hemoglobin. In addition, a significant decrease in mean reticulocyte count was noted in females only.
Bone marrow evaluation did not reveal any significant differences between control and high-dose animals (males and females) and no significant morphological alterations were noted.

There were also higher mean platelet count and prolonged prothrombin time in males only. These changes may reflect an inflammatory process such as that induced by irritation in the forestomach/stomach.

At 250 mg/kg/day
In males and females and when compared with mean controls, similar and slightly less pronounced differences were noted (i.e. decreases of red blood cell count, mean hemoglobin concentration, mean packed cell volume, mean cell volume and mean cell hemoglobin). Significantly reduced mean reticulocyte count was also recorded in females only. In view of their slight magnitude and as mean values were within or close to the range of the Historical Control Data (HCD), these findings were not considered as adverse.

At 50 mg/kg/day
In males, when compared with controls, a statistically significant decrease was observed in mean red blood cell count. As these differences were of minor magnitude, they were not considered as adverse.

The other statistically significant differences between control and test item-treated animals, namely in neutrophil and large unstained cell counts (males given 650 mg/kg/day) and in mean cell hemoglobin concentration (females given 250 mg/kg/day), were considered to be of no toxicological importance as they were of low magnitude and/or noted with no dose-relationship.

End of treatment-free period
Some differences from controls were still observed in animals treated at 650 mg/kg/day (i.e. lower mean cell volume and lower mean cell hemoglobin in males and females and lower mean hemoglobin concentration in females). These differences were noted at a lower magnitude than at the end of the treatment period, suggesting an on-going recovery for these parameters.

In high-dose males, there was a statistically significant, higher mean red blood cell count along with hemoglobin concentration and packed cell volume comparable to control values, and slightly lower reticulocyte count, and mean cell hemoglobin concentration. In high-dose females, red blood cell count and packed cell volume were similar to control values.
Red blood cell counts, packed cell volume and/or hemoglobin concentration comparable to control values and/or inside to the range of the HCD were indicative of an almost complete recovery for these parameters.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 9.
End of treatment period
At 650 mg/kg/day
In males and females, when compared with mean control values, a statistically significant increase in mean chloride and urea levels and A/G ratio of minimal magnitude were observed.
In females only, statistically significant, higher mean calcium level, along with lower mean inorganic phosphorus level, and higher mean albumin and triglyceride levels, were noted.

At 250 mg/kg/day
In males and females, when compared with mean control values, statistically significant higher mean urea levels were observed. Statistically significant differences in mean chloride, calcium, inorganic phosphorus and albumin levels and mean A/G ratio were noted in females.

At 50 mg/kg/day,
No test item treatment-related effects were observed.

The other statistically significant differences observed between control and test item-treated animals, namely in glucose (males at 50 mg/kg/day), sodium (females at 50 or 250 mg/kg/day), cholesterol (males and females at 250 mg/kg/day) and protein (females at 250 mg/kg/day) levels, and alkaline phosphatase (males at 650 mg/kg/day), aspartate aminotransferase (males and females at 650 mg/kg/day) and alanine aminotransferase (males at 250 mg/kg/day) activity were considered to be incidental, of no toxicological importance and/or not test item treatment-related as they were marginal and noted without a dose relationship and without any relationship to microscopic findings.

End of treatment-free period
Test item treatment-related effects on the blood biochemistry parameters were no longer observed in animals given 650 mg/kg/day at the end of the treatment-free period (as shown by the statistically significant, lower mean alkaline phosphatase activity in high-dose males), except for the slightly higher mean A/G ratio in females versus controls but remaining within the historical control range, suggesting almost complete reversibility.

The lower mean alkaline phosphatase activity observed in test item-treated males was considered not to be test item treatment-related as this difference was not observed at the end of the treatment period and remained within the range of physiological values.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

See Table 10.
End of treatment period
From 50 mg/kg/day
In females, when compared with mean control values, statistically significant, lower mean urine volumes along with higher mean specific gravity values were noted. However, mean urine volumes in control animals were relatively high, there was no dose response in test item-treated females and the values remained within the range of Historical Control Data. The specific gravity values, although statistically different from controls, remained within the range of the Historical Control Data. These changes were not dose-related and were observed in females only. Consequently, they were considered to be findings related to test item treatment of minor importance.

At 650 mg/kg/day
When compared with mean control values, differences were observed:
- presence of proteins was noted in 6/10 males (1 g/L) and 3/10 females (1 g/L or = 3g/L),
- moderate to marked presence of bilirubin in 7/10 females. In the absence of bilirubin increase at blood biochemistry investigations and found only in females, this was considered as probably not toxicologically relevant.

At 250 mg/kg/day,
When compared with mean control values, the presence of proteins (1 g/L) was observed in 6/10 males.

The higher mean pH value in females given 650 mg/kg/day was considered to be of no toxicological importance as it was of low magnitude, within the Historical Control Data and similar to the mean control value recorded at the end of the treatment-free period.

End of treatment-free period
Test item treatment-related effects on the urinary parameters were no longer observed in animals given 650mg/kg/day at the end of the treatment-free period.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related effects were observed during the Functional Observation Battery tests or on the motor activity data in any group.

At 650 mg/kg/day, higher mean numbers of horizontal movements and rearing were noted in males (+26% and +47% vs. controls, respectively). There were no correlating clinical signs during the study. As these differences were not statistically significant and were mainly due to the contribution of a few males, they were considered to be fortuitous and therefore of no toxicological importance.

No differences from controls were noted in the motor activity of test item-treated females. A markedly higher mean number of horizontal movements were noted in all control and test item-treated female groups. This resulted from high values recorded in the first two females in each group (between 2701 and 4520), but the cause could not be determined.

When compared with controls, a higher incidence of urination was noted in males given 50, 250 or 650 mg/kg/day (8/10, 7/10 and 7/10 animals vs. 4/10 in controls, respectively). In view of the very slight severity and in the absence of correlating clinical signs during the study, this finding was considered to be unrelated to the test item treatment.

Overall, no significant changes in autonomic, neurologic or behavioral domains were observed in male or female animals, as attested by the similar severity scores obtained by the rats given the vehicle and by those treated with test test item at 50, 250 or 650 mg/kg/day in Week 12.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See Table 11.
End of the treatment period
Test item-related changes in organ weights were seen in the kidneys from 50 mg/kg/day, and in the liver and adrenals from 250 mg/kg/day.

Kidneys
When compared with controls, test item-related minimal or slight increases in the mean absolute and relative kidney weights were found from 50 mg/kg/day in females and at 250 and 650 mg/kg/day in males. Changes were statistically significant and dose-related in magnitude. They correlated with microscopic hypertrophy/cytoplasmic alteration of tubular cells.
Changes in kidney weights at 50 mg/kg/day in males were not considered to be toxicologically significant given their low magnitude.

Liver
In the liver, there were test item-related minimal or slight increases in the mean absolute and relative weights from 250 mg/kg/day in females and at 650 mg/kg/day in males. Changes were statistically significant and dose-related in magnitude. They correlated with hepatocyte hypertrophy/cytoplasmic alteration at microscopic examination that was considered to be non-adverse.
The liver weights were also minimally higher at 50 mg/kg/day in females (reaching statistical significance for the relative weight only) and at 250 mg/kg/day in males but in the absence of correlated microscopic findings, these variations were considered not to be toxicologically significant.

Adrenals
Test item-related minimal or slight increases in the mean absolute and relative adrenal weights were seen in males at 250 and 650 mg/kg/day. This correlated with non-adverse adrenal cortex hypertrophy microscopically.
At 50 mg/kg/day in males, there were no significant changes in adrenal weights.
In females there were no relevant changes in adrenal weights at any doses.

The mean absolute and relative weights of the uterus were slightly or moderately higher (up to +40%) in females treated from 50 mg/kg/day compared to controls. These variations were considered to be related to differences in the estrous cycle and not to the test item administration.

The statistically significant increase (+10%) in brain relative weight in high-dose males was considered to be a reflection of reductions in body weight and not due to test-item-related organ toxicity.

Other differences in organ weights were minor and reflected the usual range of individual variations.

End of the treatment-free period
There was no recovery of the changes in kidney weights in animals given 650 mg/kg/day. The recovery was partial for liver weight and complete for adrenals weight.

Slight decreases in thymus weights (down to -27%, statistically significant, p<0.05) were noted in high dose females when compared to controls. In the absence of similar thymic weight variations or correlated microscopic findings at the end of the treatment period, these changes were considered to be incidental and unrelated to the test item administration.

Other differences in organ weights were minor and reflected the usual range of individual variations.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

End of the treatment period
Non-adverse test item-related gross findings were seen in the liver and stomach in males at 650 mg/kg/day.

Liver
Tan color of the liver was noted in a single high-dose male. Liver enlargement was recorded in another male from the same group. Both findings correlated with hepatocyte hypertrophy/cytoplasmic alteration at microscopic examination.

Stomach
In the stomach, although several animals from both treated and control groups showed red discoloration, this finding was considered to be toxicologically significant in a single high-dose male in which it correlated with multifocal erosions/ulcers. In other animals, this finding had no microscopic correlates.

There were no test item-related gross changes at 50 or 250 mg/kg/day.

The other macroscopic findings had no histologic correlates or correlated with common histologic findings in control rats, and were considered to be incidental.

End of the treatment-free period
There were no gross findings in the stomach and liver in animals given 650 mg/kg/day at the end of the treatment-free period, indicating complete recovery of the findings.

The kidneys were granular in 2/5 males at 650 mg/kg/day. Since there were no correlated microscopic findings, this gross change was not considered to be toxicologically significant.

The few other macroscopic findings noted at the end of the treatment period were of those commonly recorded in the rat and none were considered to be related to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See Table 12.
End of the treatment period
Test item-related microscopic changes were found in the kidneys from 650 mg/kg/day, in the liver and stomach and adrenals from 250 mg/kg/day, and in the spleen and forestomach at 650 mg/kg/day.

Kidneys
In the kidneys, non-adverse minimal or slight hypertrophy/cytoplasmic alteration of tubular cells was seen in males and females from 50 mg/kg/day. The change was characterized by enlarged cells with clear cytoplasm, and occasional basophilia and sloughed cells, and was located in the proximal tubules of the cortex. There was nothing abnormal discovered with PAS (Periodic Acid Schiff) staining (evaluated in controls and high dose animals). In a couple of females at 650 mg/kg/day, hypertrophy/cytoplasmic alteration was associated with adverse minimal tubular degeneration/necrosis affecting low numbers of tubules.
Of note, hyaline droplets were noted in the proximal tubular epithelium in 7/10 control males and were not observed in any males at 250 or 650 mg/kg/day.
At 50 mg/kg/day, non-adverse tubular hypertrophy/cytoplasmic alteration was seen in some males and females with a minimal grade, and in males was associated with lower incidence of hyaline droplets compared to controls (4/10 versus 7/10 control males).

Liver
In the liver, minimal or slight increases hypertrophy/cytoplasmic alteration was noted in a few females at 250 mg/kg/day and in most males and females at 650 mg/kg/day. This was characterized by enlarged cells with clear cytoplasm. The change was diffuse. Increased content in cytoplasmic glycogen, as shown in the increased staining by PAS (Periodic Acid Schiff) compared to controls, contributed to this change. None of these findings were considered to be adverse.
There were no test item-related microscopic changes in the liver at 50 mg/kg/day.

Spleen and liver
In the spleen and liver, the severity and/or incidence of hematopoiesis (erythropoiesis mainly) was minimally decreased in males and females at 650 mg/kg/day. This was not considered as adverse in view of their low magnitude.
There were no significant differences in the incidences and severity of hematopoiesis at 50 or 250 mg/kg/day.

Forestomach
In the forestomach, minimal or slight, diffuse or multifocal hyperplasia of the squamous epithelium was noted in 5/10 females at 650 mg/kg/day, associated with minimal or slight inflammation (mainly edema) in four of them, and with minimal focal erosion/ulcer in one of them. In males at 650 mg/kg/day, there was only minimal focal erosion/ulcer in a single individual. These erosion/ulcer were considered to be adverse at 650 mg/kg/day.
There were no test item-related microscopic findings in the forestomach at 50 or 250 mg/kg/day.

Stomach
In the stomach, minimal or slight adverse erosion/ulcer associated with hemorrhage in the mucosa was found in 2/10 males at 650 mg/kg/day. Isolated minimal hemorrhage of the gastric mucosa was also noted in an additional male and one female at 650 mg/kg/day.
At the 250 mg/kg/day, focal minimal gastric erosion/ulcer was present in one female only that was considered to be non-adverse in view of the isolated occurrence of this finding and of its low severity.

There were no test item-related changes in the stomach at 50 mg/kg/day.

Adrenals
In the adrenals, minimal diffuse hypertrophy of the cortex was noted in 3/10 males at 250 mg/kg/day and 4/10 males at 650 mg/kg/day. This finding was considered to be non-adverse in view of the minimal severity and of absence of degeneration/necrosis and may have been related to stress in these animals.
There were no changes in the adrenals from males at 50 mg/kg/day.
There were no test item-related changes in females at any doses.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the rat.

End of the treatment-free period
In the kidneys, adrenals, forestomach and stomach, there were no relevant microscopic changes in any recovery animals examined, indicating complete recovery in both sexes.

In the liver, a single high-dose male showed minimal hypertrophy/cytoplasmic alteration of the hepatocytes.

Regarding the hematopoiesis in the liver and spleen, recovery was complete except in the spleen of males. Slight hematopoiesis was still observed in the spleen from test item-treated males while moderate hematopoiesis was seen in controls, suggesting minimally lower grades in test item treated males compared to controls.

Histopathological findings: neoplastic:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: gastrointestinal tract and urinary

Organ:

kidney

stomach

other: forestomach

Treatment related:

yes

Dose response relationship:

yes

Table 5: Clinical signs

 

Sex	Male				Female
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
Ptyalism	-	-	-	3	-	-	-	2
Reflux at administration	2	-	-	1	1	-	4	3
Total number of affected animals	2/15	0/10	0/10	4/15	1/15	0/9	4/10	5/15

-: no clinical signs.

Table 6: Body weight

 

Sex	Male				Female
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
Treatment period
Mean BW gain Days 1/29	+158	+154	+157	+145	+71	+65	+74	+71
Mean BW gain Days 29/57	+64	+66	+69	+56	+34	+31	+28*	+27**
Mean BW gain Days 57/91	+40	+39	+35	+30*	+17	+19	+11	+3**
Mean BW gain Days 1/91	+262	+260	+262	+232*	+123	+117	+112	+101**
% from controls	-	-1	0	-11	-	-5	-9	-18
Mean body weight on Day 1	172	170	171	171	121	120	117	125
Mean body weight on Day 91	434	429	432	403	244	237	230	226*
% from controls	-	-1	0	-7	-	-3	-6	-7
Treatment-free period
Mean BW gain Days 91/133	+27	-	-	+41*	+5	-	-	+12*
Mean BW on Day 133	468	-	-	458	247	-	-	238
% from controls	-	-	-	-2	-	-	-	-4

Statistically significant from controls: *: p<0.05; **: p<0.01; -: not applicable.

Table 7: Food consumption

Sex	Male				Female
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
Treatment period
Days 29 to 36	28.0	26.6	27.1	25.1*	18.8	17.9	17.3	17.8
Days 36 to 43	27.8	27.0	26.6	22.1*	19.2	17.4	17.4	18.0
Days 57 to 64	26.9	26.8	26.6	24.4*	19.3	17.6*	18.5	17.5*
Days 78 to 85	25.7	25.3	25.4	23.5	18.0	16.3	16.8	16.0*

Statistically significant from controls: *: p<0.05.

Table 8: Haematology

 

Sex	Male
Dose level (mg/kg/day)	0	50	250	650
End of treatment period
Red blood cell count (T/L)	9.22 (7.69-10.73)	8.77* -5%	8.66** -6%	8.01** -13%
Hemoglobin (g/dL)	16.2 (14.5-17.7)	15.6 -4%	14.4** -11%	12.8** -21%
Packed cell volume (L/L)	0.47 (0.42-0.55)	0.45 -4%	0.42** -11%	0.38** -19%
Mean cell volume (fl)	50.7 (48.1-55.9)	51.7 +2%	49.1 -3%	47.3** -7%
Mean cell hemoglobin (pg)	17.6 (16.1-19.6)	17.8 +1%	16.7* -5%	16.1** -9%
Platelet count (G/L)	726 (282-864)	804 +11%	819 +13%	959** +32%
Reticulocyte count (T/L)	0.24 (na)	0.21 -13%	0.17 -29%	0.18 -25%
Prothrombin time (s)	21.8 (18.4-25.6)	20.6 -6%	23.5 +8%	24.4* +12%
End of treatment-free period
Red blood cell count (T/L)	8.82	/	/	9.60* +9%
Hemoglobin (g/dL)	14.9	/	/	15.0 +1%
Packed cell volume (L/L)	0.44	/	/	0.46 +5%
Mean cell volume (fl)	50.4	/	/	48.0* -5%
Mean cell hemoglobin (pg)	16.9	/	/	15.6* -8%
Mean cell hemoglobin concentration (g/dL)	33.5	/	/	32.5* -3%
Platelet count (G/L)	717	/	/	753 +5%
Reticulocyte count (T/L)	0.25	/	/	0.16 -36%
Prothrombin time (s)	18.2	/	/	18.7 +3%
Sex	Female
Dose level (mg/kg/day)	0	50	250	650
End of treatment period
Red blood cell count (T/L)	8.36 (7.43-8.97)	8.17 -2%	7.94* -5%	7.18** -14%
Hemoglobin (g/dL)	15.9 (14.3-16.6)	15.5 -3%	14.0** -12%	12.7** -20%
Packed cell volume (L/L)	0.45 (0.41-0.49)	0.44 -2%	0.41** -9%	0.37** -18%
Mean cell volume (fl)	53.9 (52.0-59.4)	53.9 0%	51.7** -4%	50.8** -6%
Mean cell hemoglobin (pg)	19.0 (17.1-20.5)	19.0 0%	17.6** -7%	17.7** -7%
Reticulocyte count (T/L)	0.19 (na)	0.16 -16%	0.14* -26%	0.12** -37%
End of treatment-free period
Red blood cell count (T/L)	8.08	/	/	8.25 +2%
Hemoglobin (g/dL)	14.7	/	/	14.2* -3%
Packed cell volume (L/L)	0.43	/	/	0.42 -2%
Mean cell volume (fl)	53.7	/	/	51.6** -4%
Mean cell hemoglobin (pg)	18.2	/	/	17.2** -5%
Reticulocyte count (T/L)	0.16	/	/	0.15 -6%

/: not applicable; statistically significantfrom controls:*: p<0.05 and**: p<0.01.

%: percentage differencevs.controls; ( )    : minimum-maximum values from Historical Control Data (HCD).

Bold = treatment-related.

Table 9: Blood biochemistry

 

Sex		Male
Dose level (mg/kg/day)		0		50		250		650
End of treatment period
Chloride (mmol/L)		106.0 (100.4-107.4)		106.1 0%		106.9 +1%		108.6** +2%
Urea (mmol/L)		5.0 (3.9-6.4)		4.9 -2%		6.0** +20%		5.9** +18%
A/G ratio		1.41 (1.34-1.67)		1.42 +1%		1.45 +3%		1.54** +9%
End of treatment-free period
Chloride (mmol/L)		106.7		/		/		106.1 -1%
Urea (mmol/L)		4.6		/		/		4.7 +2%
A/G ratio		1.35		/		/		1.32 -2%
Sex	Female
Dose level (mg/kg/day)	0		50		250		650
End of treatment period
Chloride (mmol/L)	109.2 (100.5-111.8)		109.8 +1%		113.2** +4%		112.6** +3%
Calcium (mmol/L)	2.50 (2.38-2.69)		2.55 +2%		2.62** +5%		2.66** +6%
Inorganic phosphorus (mmol/L)	1.59 (1.23-2.03)		1.40 -12%		1.31* -18%		1.31* -18%
Urea (mmol/L)	5.5 (4.9-7.1)		6.1 +11%		6.9** +25%		8.4** +53%
Albumin (g/L)	38 (36-42)		40 +5%		43** +13%		42** +11%
A/G ratio	1.51 (1.41-1.70)		1.57 +4%		1.61** +7%		1.71** +13%
Triglycerides (mmol/L)	0.27 (0.18-0.61)		0.32 +19%		0.29 +7%		0.39** +44%
End of treatment-free period
Chloride (mmol/L)	107.6		/		/		105.9 -2%
Calcium (mmol/L)	2.51		/		/		2.58 +3%
Inorganic phosphorus (mmol/L)	1.29		/		/		1.24 -4%
Urea (mmol/L)	6.6		/		/		7.0 +6%
Albumin (g/L)	38		/		/		42 +11%
A/G ratio	1.44		/		/		1.56** +8%
Triglycerides (mmol/L)	0.39		/		/		0.49 +26%

/: not applicable; statistically significantfrom controls:*: p<0.05 and**: p<0.01.

%: percentage differencevs.controls; ( )    : minimum-maximum values from Historical Control Data (HCD).

Bold = treatment-related

Table 10: Urinalysis

 

Sex	Male				Female
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
End of treatment period
Volume (mL)	11	11 0%	8 -27%	8 -27%	13 (3-27)	5** -62%	6** -54%	4** -69%
Specific gravity	1027	1029 0%	1036 +1%	1036 +1%	1020 (1010-1045)	1038** +2%	1033 +1%	1039** +2%
pH	7.6	7.5 -1%	7.8 +3%	7.8 +3%	6.1 (5.5-7.0)	6.1 0%	6.3 +3%	7.0** +15%
Presence of proteins
. 0.3 g/L	9	4	3	2	1	4	4	3
. 1 g/L	0	2	6	6	0	2	0	1
. = 3 g/L	0	0	0	0	0	0	0	2
Presence of bilirubin
. low	0	0	0	0	0	0	1	1
. moderate	0	0	0	0	0	0	0	4
. high	0	0	0	0	0	0	0	3
End of treatment-free period
Volume (mL)	10	/	/	9	8	/	/	6
Specific gravity	1030	/	/	1029	1028	/	/	1029
pH	7.1	/	/	7.4	7.0	/	/	6.8
Presence of proteins
. 0.3 g/L	4	/	/	3	0	/	/	2
Presence of bilirubin
. low	1	/	/	0	0	/	/	2

/: not applicable; statistically significantfrom controls:**: p<0.01.

%: percentage differencevs.controls; ( )    : minimum-maximum values from Historical Control Data (HCD).

Bold = treatment-related.

Table 11: Organ weights

Relevant changes in mean absolute and relative organ weights in treated groups at the end of the treatment period (% changes from controls)

 

Sex	Male			Female
Group	2	3	4	2	3	4
Dose level (mg/kg/day)	50	250	650	50	250	650
Exam. animals / Num. of animals	10/10	10/10	10/10	9/10	10/10	10/10
- Final body weight	0	+1	-8	-2	-4	-8
- Kidneys
. absolute	+5	+13**	+25**	+11	+16*	+25**
. relative	+5	+12**	+35**	+14*	+22**	+35**
- Liver
. absolute	-2	+13	+23**	+8	+21	+34**
. relative	-2	+12	+33**	+11*	+27**	+46**
- Adrenal glands
. absolute	-2	+19*	+22*	+4	-3	-3
. relative	-2	+18	+32**	+7	+2	+5

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Relevant changes in mean absolute and relative organ weights in high-dose group at the end of the treatment-free period (% changes from controls)

Sex	Male	Female
Group	4	4
Dose level (mg/kg/day)	650	650
Exam. animals / Num. of animals	5/5	5/5
- Final body weight	-1	-4
- Kidneys
. absolute	+27*	+30**
. relative	+27**	+35**
- Liver
. absolute	+18	+13
. relative	+18**	+17**

Statistically significant from controls: *: p<0.05, **: p<0.01.

Statistical significance determined for organ weights values and not percent changes.

Table 12: Microscopic examination

Incidence and severity of selected microscopic changes in the kidneys

at the end of the treatment-period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
No. animals	10	10	10	10	10	9	10	10
Kidneys
- Hypertrophy/cytoplasmic  alteration; tubule
Minimal (grade 1)	-	7	6	7	-	5	5	4
Slight (grade 2)	-	-	3	2	-	-	5	6
- Degeneration/necrosis;  tubule
Minimal (grade 1)	-	-	-	-	-	-	-	2
- Hyaline droplets; tubular  epithelium
Minimal (grade 1)	5	4	-	-	-	-	-	-
Slight (grade 2)	2	-	-	-	-	-	-	-

-: no findings.

 

Incidence and severity of hepatocyte hypertrophy/cytoplasmic alteration in the liver

at the end of the treatment-period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
No. animals	10	10	10	10	10	9	10	10
Liver
- Hypertrophy/cytoplasmic alteration; hepatocyte
Minimal (grade 1)	-	-	-	8	-	-	2	7
Slight (grade 2)	-	-	-	1	-	-	1	1
- PAS staining
Minimal (grade 1)	7	NA	NA	5	1	NA	NA	3
Slight (grade 2)	-	NA	NA	4	1	NA	NA	5
Moderate (grade 3)	-	NA	NA	1	-	NA	NA	-

-: no findings.NA: Not Applicable (special stain not evaluated).

 

Incidence and severity of hematopoiesis in the spleen and liver

at the end of the treatment-period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
No. animals	10	10	10	10	10	9	10	10
Spleen
Minimal (grade 1)	2	1	1	9	-	-	-	3
Slight (grade 2)	4	3	6	-	1	1	2	5
Moderate (grade 3)	4	6	3	1	9	7	8	2
Marked (grade 4)	-	-	-	-	-	1	-	-
Liver
Minimal (grade 1)	7	6	5	4	6	7	8	9
Slight (grade 2)	-	-	-	-	4	2	2	-

-: no findings.

 

Incidence and severity of selected microscopic findings in the forestomach and stomach

at the end of the treatment-period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
No. animals	10	10	10	10	10	9	10	10
Forestomach
- Hyperplasia; squamous cell
Minimal (grade 1)	-	-	-	-	-	-	-	3
Slight (grade 2)	-	-	-	-	-	-	-	2
- Inflammation
Minimal (grade 1)	-	-	-	-	-	-	-	3
Slight (grade 2)	-	-	-	-	-	-	-	1
- Erosion/ulcer
Minimal (grade 1)	-	-	-	1	-	-	-	1
Stomach
- Erosion/ulcer
Minimal (grade 1)	-	-	-	1	-	-	1	-
Slight (grade 2)	-	-	-	1	-	-	-	-
- Hemorrhage
Minimal (grade 1)	-	-	-	3	-	-	-	1
Total of affected animals (forestomach and/or stomach)	0	0	0	4	0	0	1	6

-: no findings.

 

Incidence and severity of adrenal cortex hypertrophy at the end of the treatment-period

 

Sex	Male				Female
Group	1	2	3	4	1	2	3	4
Dose level (mg/kg/day)	0	50	250	650	0	50	250	650
No. animals	10	10	10	10	10	9	10	10
Adrenals
- Hypertrophy; cortex
Minimal (grade 1)	-	-	3	4	-	NA	NA	-

-: no findings. NA: Not Applicable.

 

Incidence and severity of relevant microscopic changes in the liver and spleen in males

at the end of the treatment-free period (n = 5)

 

Group	1	4
Dose level (mg/kg/day)	0	650
Liver
- Hypertrophy/cytoplasmic alteration; hepatocyte
Minimal (grade 1)	-	1
Spleen
- Hematopoiesis
Slight (grade 2)	-	5
Moderate (grade 3)	5	-

-: no findings.

Conclusions:

The toxicity of the test item was evaluated after daily oral administration (gavage) to Wistar rats at the dose levels of 50, 250 and 650 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period.

Under the experimental conditions of the study, the NOAEL (No Observed Adverse Effect Level) was established at 250 mg/kg/day based on multiple correlated effects observed at 650 mg/kg/day indicative of an impact on red blood cell mass and adverse microscopic lesions in several organs (erosions/ulcers in the forestomach and/or stomach in both sexes and tubular degeneration/necrosis in kidneys).

Executive summary:

The objective of this study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks. On completion of the treatment period, designated animals were held for a 6-week treatment-free period in order to evaluate the reversibility of any findings.

This GLP study was carried out according to OECD test guideline No. 408 (21 September 1998).

 

Methods

One group of 15 male and 15 female Wistar rats was treated daily by the oral route (gavage) with the test item at the dose level of 650 mg/kg/day (group 4) for 13 weeks. Two other groups of 10 males and 10 females were treated with the test item at dose levels of 50 and 250 mg/kg/day (groups 2 and 3, respectively). One group of 15 males and 15 females received the vehicle only (drinking water treated by reverse osmosis) under the same experimental conditions and acted as a control group (group 1). A constant dosage volume of 10 mL/kg/day was used. At the end of the treatment period, the animals were sacrificed, except for the first five group 1 and 4 animals per sex, which were kept for a 6-week treatment-free period. Dosing formulations were prepared using a correction factor of 1.24 and the pH was measured on Day 1.

The actual test item concentrations in the dosing formulations were determined in Weeks 1, 4, 8 and 11.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly and a Functional Observation Battery (FOB) was conducted in Week 12.

Body weight was recorded pre-test, on Day 1 and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals (pre-test) and on control and high-dose animals (Week 13). Hematology, blood biochemistry and urinary investigations were performed at the end of the treatment and treatment-free periods.

On completion of the treatment or treatment-free period, the animals were sacrificed and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on groups 1 and 4 animals sacrificed at the end of the treatment period, on the group 2 animal which was found dead, and on all macroscopic lesions from group 2 and 3 animals sacrificed on completion of the treatment period. The liver and kidneys of the group 1 and 4 animals sacrificed at the end of the treatment period were examined after PAS (Periodic Acid Schiff) staining. Selected tissues, namely the liver, kidneys, stomach (with forestomach) and spleen from group 2 and 3 animals euthanized at the end of the treatment period and from recovery animals, as well as the adrenals from group 2 and 3 males sacrificed at the end of the treatment period and from recovery males were also microscopically examined.

Results

 

Actual concentrations of the test item in the dosing formulations administered to the animals during the study remained within an acceptable range (-3.4% to +9.5%) compared to the nominal concentrations.

 

At 650 mg/kg/day

The value of pH measured in the dosing formulation on Day 1 was 12.1.

The following test item treatment-related adverse findings were observed:

An adverse moderate decrease in red blood cell parameters was noted including red blood cell counts, hemoglobin concentration, packed cell volume, mean cell volume and mean cell hemoglobin in males and females. These changes were noted along with thrombocytosis and prolonged prothrombin time in males or with lower reticulocyte count in females. Some of these changes were not reversible. These hematological changes correlated with decreased hematopoiesis (mainly erythropoiesis) seen only at 650 mg/kg/day in the spleen and to a lesser extent in the liver.

 

The following test item treatment-related non-adverse findings were observed:

Slightly/moderately lower body weight gain was recorded throughout the treatment period in males and females, resulting in minimally lower body weights on completion of the treatment period and correlated with slight food consumption. This effect which was considered to be non adverse was recovered at the end of the treatment free period.

Blood biochemistry changes were considered as non-adverse and included a slight increase in chloride concentration and A/G ratio and marked higher urea level in males and females. Slight higher calcium, albumin and triglyceride levels and lower inorganic phosphorus level were noted in females only. At the end of the treatment-free period, slightly higher mean A/G ratio was still observed in females.

Urinalysis changes, which were considered as non-adverse and were fully recovered at the end of the treatment-free period, consisted of lower volume and higher specific gravity in females, moderate/marked proteinuria in males and females and bilirubinuria in females.

 

The test item administration induced microscopic changes in the kidneys, liver, forestomach, stomach, spleen and adrenals:

- In the kidneys, reversible tubular hypertrophy/cytoplasmic alteration correlated with increased kidney weights. This non-adverse finding was associated with minimal, adverse tubular degeneration/necrosis in 2/10 females.

- In the liver, hepatocyte hypertrophy/cytoplasmic alteration correlated with increased liver weights. This non-adverse finding was partially reversible in males and totally reversible in females.

- In the forestomach and stomach, the observed findings including erosion/ulcer considered as adverse, squamous epithelial hyperplasia, inflammation and hemorrhage were suggestive of an irritant effect of the test item on the gastric epithelium and considered as adverse. These findings were totally reversible after a 6-week treatment-free period.

- In the spleen and to a lesser extent in the liver, there was non-adverse decreased hematopoiesis (mainly erythropoiesis). This finding was partially reversible in males and totally reversible in females.

- In the adrenals, non-adverse reversible, minimal cortex hypertrophy correlated with increased adrenal weights.

At 250 mg/kg/day

The value of pH measured in the dosing formulation on Day 1 was 11.9.

The following test item treatment-related non-adverse findings were observed:

Slightly but non-adverse lower body weight gain was recorded throughout the second month of the treatment period in females.

A non-adverse slight decrease in red blood cell parameters was noted, including red blood cell count, hemoglobin concentration, packed cell volume, mean cell volume and mean cell hemoglobin in males and females. These changes were noted along with lower reticulocyte count in females.

Higher urea level was noted in males and females. A slight increase in chloride concentration and A/G ratio, slightly higher calcium and albumin levels and lower inorganic phosphorus level were noted in females only. None of these findings were considered to be adverse.

Urinalysis changes were considered to be non-adverse and consisted of lower volume and higher specific gravity in females and moderate proteinuria in males.

The test item administration induced non-adverse microscopic changes in the kidneys, liver, forestomach, stomach, and adrenals.

- In the kidneys, tubular hypertrophy/cytoplasmic alteration correlated with increased kidney weights.

- In the liver, hepatocyte hypertrophy/cytoplasmic alteration was seen in a few females only and correlated with increased liver weights.

- In the stomach, minimal erosion/ulcer of a single animal was observed but not considered to be adverse.

- In the adrenals, minimal cortex hypertrophy correlated with increased adrenal weights in males.

 

At 50 mg/kg/day

The value of pH measured in the dosing formulation on Day 1 was 11.5.

No test item treatment-related effects were observed in any of the evaluated parameters.

Non-adverse slightly lower red blood cell counts were observed in males only.

The only test item-related microscopic changes were seen in the kidneys and consisted of non-adverse tubular hypertrophy/cytoplasmic alteration correlated with increased kidney weights.

 

Conclusion

The toxicity of the test item was evaluated after daily oral administration (gavage) to Wistar rats at the dose levels of 50, 250 and 650 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period.

 

Under the experimental conditions of the study, the NOAEL (No Observed Adverse Effect Level) was established at 250 mg/kg/day based on multiple correlated effects observed at 650 mg/kg/day indicative of an impact on red blood cell mass and adverse microscopic lesions in several organs (erosions/ulcers in the forestomach and/or stomach in both sexes and tubular degeneration/necrosis in kidneys).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Study complete and sufficient to fulfill the endpoint requirements.

System:

haematopoietic

Organ:

kidney

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

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).

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

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study performed according to the OECD guideline 422 and GLP compliant

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: other

Justification for classification or non-classification

Reduced locomotor activity, reduced body weight gain for male animals, changes in haematological and clinical biochemistry parameters, organ weight changes, and treatment related macroscopic and microscopic changes in the stomach were observed at the dose level of 1000 mg/kg bw/d. Most findings recovered during the 14-day treatment free period. The No Observed Adverse Effect Level (NOAEL) was 300 mg/kg bw/d. Therefore, according to the CLP and DSD criteria, no classification is warranted for the repeated toxicity.