3-aminopropyldimethylamine

Endpoint summary

Description of key information

Repeated dose toxicity: oral
In a 28 days repeated dose oral toxicity study conducted on rats, the NOAEL was 50 mg/kg bw/day and the LOAEL was found to be 250 mg/kg bw.

 

In a 90 days repeated dose oral toxicity study with the structurally related Diethylaminopropylamine (as pH-neutralized dose formulations) conducted on rats, the NOAEL was established at 750 mg/kg/day in males and 250 mg/kg/day in females.

 

 

 

 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The basic structures of DMAPA and DEAPA are the same: lower aliphatic diamines with a tertiary and a primary amino functionality. Only the two alkyl residues at the tertiary nitrogen atom vary: DEAPA consists of two ethyl groups instead of two methyl groups at DMAPA. The comparison of the physicochemical data shows that the properties of the substances are similar. In general, based on the physicochemical properties and the experimental animal data, target and source substance are expected to become bioavailable via all routes.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
No impurities were identified that would impact the toxicological profile of DEAPA or DMAPA.

3. ANALOGUE APPROACH JUSTIFICATION
Please refere to WoE/read-across justification in chapter 13.

4. DATA MATRIX
(1)No compound related gross findings were observed in terminally killed animals of all dose groups in the available 28-day study with DMAPA. The four high dose female rats which died intercurrently showed macroscopically visible changes such as discoloration of lungs with multiple red spots on its surfaces and foamy content. One of them also showed a small spleen. Histopathological examinations revealed lesions in the four females of the high dose group dying intercurrently. These lesions included congestion of organs, pulmonary hemorrhage, and edema, consistent with cardiorespiratory failure as cause of death. In addition, one of these females exhibited marked loss of lymphatic follicles of the spleen with massive marginal zone and periarteriolar lymphoid sheath atrophy. In the one high dose male rat which had shown clinical signs, focal ballooning degeneration of the squamous epithelium of the forestomach was found. In conclusion 3-Dimethylaminopropylamin caused clinical symptoms in male and mortality in female Wistar rats when administered 28 times during 29 days at the dose level of 250 mg/kg body weight. The female rats seem to be more sensitive.

(2) Results of the performed exposure assessment cover all relevant exposures throughout the life cycle of the substance DMAPA and demonstrate the absence of significant exposure in all scenarios of the manufacture and all identified uses as referred to in Annex VI section 3.5. The use of the substance is restricted to professional and industrial use. Workers can at maximum be exposed acutely and short-term as the substance is used mainly in closed systems. Since DMAPA is classified as skin corrosive Cat. 1B (H314), skin sensitizer Cat. 1B (H317) and respiratory irritant (H335, STOT SE Cat. 3) a qualitative risk assessment for the local effects is conducted and appropriate risk management measures are implemented to control these risks.

(3) DNELs for longterm-exposure have been derived from results of a 28-days repeated Dose Toxicity Study (OECD 407). The derived DNEL is relevant and appropriate both to the information requirement to be omitted and for risk assessment purposes. Furthermore, as the derived DNEL for DMAPA is below the values of other comparable tertiary Alkylamines, it is considered sufficiently low to protect from local effects of DMAPA representing a precautionary approach.

(4) The comparison of the derived DNEL with the results of the exposure assessment shows that exposures are always well below the derived DNEL. Thus the industrial use is safe for the worker and there is no chronic exposure. The combined risk characterization ratios (RCR combined) are < 1 (please compare chapter 9, CSR for the single values);

(5)Taken from the OECD SIDS: “…Conclusion: With regard to the 28-subacute oral toxicity study with male and female Wistar rats a no-observed-adverse-effect-level (NOAEL) of 50 mg/kg body weight was established. Recommendation: no need for follow-up test; Priority setting: Low priority or concern.” The cause of death in the high dose group is likely be related to the corrosiveness of the substance. As no other systemic toxicity and effects on organs were observed, further testing with prolonged exposure is not thought to deliver additional information. In addition, there are subchronic and chronic oral toxicity studies available with the structurally related substance Diethylaminopropylamine (DEAPA). In a 90 days repeated dose oral toxicity study with DEAPA (as pH-neutralized dose formulations) conducted on rats, the NOAEL was established at 750 mg/kg/day in males and 250 mg/kg/day in females.
Taking into account the evidences from these studies it can be assumed that a prolonged treatment period with DMAPA will not provide further information on the hazard endpoint repeated dose toxicity. Therefore, there is no need for a further repeated dose 90 day toxicity study with DMAPA.
For more details please refer to WoE/read-across justification in chapter 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

no

Conclusions:

The toxicity of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 250 or 750 mg/kg/day for 13 weeks followed by a 6-week treatment-free period. Under the experimental conditions of the study, clinical signs of poor condition were observed at the dose level of 750 mg/kg/day in four females, which induced the premature sacrifice of one of them more severely affected.No other adverse effects were observed in the study.Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 750 mg/kg/day in males and 250 mg/kg/day in females.

Executive summary:

Based on the physicochemical properties and the experimental animal data, target and source substance are expected to have the same toxicological profile. There were no indications of a specific target organ toxicity in the repeated dose studies with DMAPA and DEAPA. Therefore,no classification is warranted for repeated dose toxicity according to CLP criteria.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 August 2015 - 28 January 2016

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: on the first day of treatment, the animals were 9 weeks old.
- Weight at study initiation: the males had a mean body weight of 425 g (range: 383 g to 479 g) and the females had a mean body weight of 265 g (range: 234 g to 300 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes of the same sex and in the same group, in polycarbonate cages with stainless steel lids
- Diet (e.g. ad libitum): SSNIFF R/M-H pelleted maintenance diet
- Water (e.g. ad libitum): tap water (filtered with a 0.22 µm filter)
- Acclimation period: 35 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: From: 17 September 2015 To: 28 January 2016

Route of administration:

oral: gavage

Vehicle:

other:

Remarks:

Drinking water treated by reverse osmosis

Details on oral exposure:

TYPE OF FORMULATION:
solution in the vehicle.

PREPARATION OF DOSING SOLUTIONS:
- Preparation procedure:
The test item was weighed and mixed with the required quantity of vehicle. The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of hydrochloric acid (HCl 5N, VWR Prolabo®); the volume of HCl added was recorded for each concentration. The final pH was checked and recorded by using a pH-meter.
- Frequency of preparation and storage conditions:
Fresh dose formulations were prepared on the day of administration and kept at room temperature prior to administration.

VEHICLE
- Concentration in vehicle: 10, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gas Chromatography with FID detection (GC-FID).

Duration of treatment / exposure:

13 weeks.

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.:

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

No. of animals per sex per dose:

Groups 1 and 4: 15 males and 15 females
Groups 2 and 3: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The dose-levels were selected based on the results of a previous toxicology study performed in the same species. Male and female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations) daily, by gavage, at dose-levels of 100, 300 or 1000 mg/kg/day for 2 weeks. At 1000 mg/kg/day, in-life observations were limited to ptyalism in both sexes, and lower body weight gain at treatment initiation resulted in a lower final body weight in males. Histopathology findings consisted of minimal to slight hyperplasia of squamous cells, associated with minimal to slight hyperkeratosis within the forestomach in all males and females given 1000 mg/kg/day.Thus, based on these available data, the following dose-levels were selected for the present study: 50, 250 and 750 mg/kg/day.

- Post-exposure recovery period in satellite groups: 6 weeks

Observations and examinations performed and frequency:

MORBIDITY AND MORTALITY: Yes
- 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, including weekends and public holidays.

CLINICAL OBSERVATIONS: Yes
- Time schedule: each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL EXAMINATION: Yes
- Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then at least once a week until the end of the study.

FUNCTIONAL OBSERVATION BATTERY: Yes
- Time schedule: all main animals were evaluated once in Week 13 (before the daily treatment). As no relevant changes were observed at the end of the treatment period, FOB examinations were not carried out at the end of the treatment-free period.

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

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

FOOD CONSUMPTION: Yes
- Time schedule: The quantity of food consumed by the animals in each cage was recorded at least once a week, over a 6 or 7-day period, during the study.Food consumption was calculated per animal and per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examinations were performed on all animals, before the beginning of the treatment period, on all main animals on one occasion at the end of the treatment period.As no relevant changes were detected in the treated animals at the end of the treatment period, the recovery animals were not examined at the end of the treatment-free period.

MONITORING OF ESTROUS CYCLE: Yes
The estrous cycle stage was determined for each female sacrificed at the end of the treatment period, from a fresh daily vaginal lavage (stained with methylene blue) for 21 consecutive days before the end of the treatment period.In view of the findings observed at the end of the treatment period, these examinations were carried out daily for 5 consecutive days before the end of the treatment-free period.

HAEMATOLOGY: Yes
- Time schedule for peripheral blood: the parameters were determined for all surviving animals sacrificed at the end of the treatment period.
- Time schedule for bone marrow: two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals sacrificed on completion of the treatment or treatment-free period.In view of the findings observed at the end of the treatment period, these examinations were also carried out at the end of the treatment-free period.

CLINICAL CHEMISTRY: Yes
- Time schedule: the parameters were determined for all surviving animals sacrificed at the end of the treatment period. In view of the findings observed at the end of the treatment period, these examinations were also carried out at the end of the treatment-free period.

URINALYSIS: Yes
- Time schedule: the parameters were determined for all surviving animals sacrificed at the end of the treatment period.In view of the findings observed at the end of the treatment period, these examinations were also carried out at the end of the treatment-free period.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: all main animals were evaluated once in Week 13 (before the daily treatment).
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity.

SEMINOLOGY: Yes
- Time schedule: before sacrifice at the end of the treatment period, each male was anesthetized by an intraperitoneal injection of sodium pentobarbital.As no relevant treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.
- Epididymal sperm
Sperm from the cauda epididymis was sampled for motility and morphology investigations.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.
- Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.
- Epididymal sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
. normal,
. normally shaped head separated from flagellum,
. abnormal head separated from flagellum,
. abnormal head with normal flagellum,
. abnormal head with abnormal flagellum,
. normally shaped head with abnormal flagellum.
. Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.
- Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed (without the albuginea) and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature
spermatozoa) were counted in a microscope slide counting chamber. Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (a time divisor of 6.10 which represents the duration of spermatogenic cycle of homogenization-resistant testicular spermatids).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

ORGAN WEIGHTS: Yes
The body weight of each animal was recorded before sacrifice. 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 sacrifice) was calculated.

HISTOPATHOLOGY: Yes
A microscopic examination was performed on:
- control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and one prematurely sacrificed female (group 4),
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period. According to microscopic findings observed in the control and high-dose animals (groups 1 and 4), the following tissues from low- and intermediate-dose animals (groups 2 and 3) and from the recovery animals (groups 1 and 4) were examined: brain, forestomach, gut-associated lymphoid tissue, kidneys, mesenteric lymph nodes (males only), pituitary gland, spleen and thymus.

Statistics:

Citox software was used to perform the statistical analyses of body weight, food consumption, hematology, blood biochemistry and urinalysis data. PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, signs of poor clinical condition (thin appearance, hunched posture, piloerection, bent head, loud and/or abdominal breathing, and/or dyspnea) were noted in 1/15 males for few days and 3/14 females from Week 11. Thin appearance, hunched posture, dyspnea, abdominal, loud breathing and/or bent head were considered to be adverse at this dose-level in 3/14 females (one out of three females also showing a large chronic hematoma in the cranium at microscopic examination) as these signs were not just transient in contrast to males. Red discoloration of urine or vagina was transiently noted in 1/15 males and 2/14 females together with soiled urogenital region in one female.At 250 mg/kg/day, loud breathing (in 2/10 males) and red discoloration of the vagina (in 1/10 females) were observed.At 50 mg/kg/day, hunched posture and piloerection were noted in 1/10 females for 2 days in Week 13.These signs were not dose-related. They were considered to be of minor toxicological significance as they were sporadically observed at these dose-levels.Ptyalism was transiently observed in 12/15 males and 14/14 females given 750 mg/kg/day (generally from Week 2 or 3) and in 1/15 males and 2/15 females given 250 mg/kg/day (between Weeks 11 and 13). Reflux at dosing noted in 2/15 males and 2/15 females on one occasion at the high dose-level was considered to be test item treatment-related.These signs, commonly observed when a test item is administered by gavage, were considered not to represent an adverse effect.The other clinical signs recorded during the study, i.e. alopecia, scabs, soiled nose and mouth, soiled head and neck, wound, thinning of hair, opacity of eyes, bent tail, nodosities, chromodacryorrhea and/or chromorhynorrhea were observed both in control and test item-treated animals with no dose-relationship. They were therefore considered to be unrelated to the test item treatment.Test item treatment-related clinical signs were no longer observed over the treatment-free period.In follow-up to a wound on the neck region of one group 2 male, an antiseptic solution (povidone iodine, Vetedine®), was applied once or twice a day for 12 days. See table 1.

Mortality:

mortality observed, treatment-related

Description (incidence):

One femake given 750 mg/kg/day was sacrificed for ethical reasons on Day 72 (Week 11). Prior to sacrifice, thin appearance, piloerection, hunched posture, ptyalism, loud breathing, hypoactivity and coldness to the touch were observed. A body weight loss (-53 g, i.e. -20% of its Day 1 body weight) was recorded between Days 1 and 71, along with a low mean food consumption in the cage (up to -28% when compared to controls).At necropsy, the spleen was enlarged, the stomach was distended with food and the stomach wall showed a red discoloration of approximately 0.2 cm in diameter. The thymus showed red discoloration of up to 0.5 cm in diameter. Test item-related microscopic vacuoles were observed in the white matter from the brain (hippocampus, cingulum, cerebral peduncle and cerebellum), in the pars nervosa of the pituitary gland, in the renal tubules, in the choroid plexus, in the spleen and in the GALT.These vacuoles in brain, kidneys and pars nervosa were considered to have probably contributed to the moribundity of this animal.In addition, the following lesions which may be related partly with stress were noted: atrophy of the ovaries, uterus and vagina, pancreas degranulation, increased adipose tissue in the bone marrow associated with decreased cellularity of the hematopoietic cells and thymus atrophy. This unscheduled death was considered to be probably test item treatment-related.No other unscheduled deaths were observed during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day in males, when compared with controls, lower mean body weight gain was recorded throughout the treatment period (-21% vs. controls). This was statistically significant during the first and the third months (p<0.01 and p<0.05, respectively), leading to a slightly, non-statistically significant, lower mean body weight on completion of the treatment period (-6% vs. controls). This effect was attributed to the test item treatment, but considered as non-adverse. During the recovery period, a higher mean body weight gain was observed in males previously treated with 750 mg/kg/day, leading to a terminal mean body weight similar to the control animals.At 50 and 250 mg/kg/day in males, no relevant effects were noted on body weight or body weight gain during the study period.At all dose-levels of the test item in females, some instances of lower mean body weight gain, statistically significant on few occasions, were reported compared with controls over the treatment or treatment-free period. As these differences were occasional, not dose-related and/or of minimal amplitude, and did not affect the mean body weight, they were considered to be of no toxicological importance. Slight mean body weight loss (-5 g vs. +3 g in controls) was also recorded in females previously given 750 mg/kg/day during the last 2 weeks of the treatment-free period. This was considered to be fortuitous. See table 2.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In females, transient statistically significant lower mean food consumption, when compared with controls, was noted in the fourth week of treatment at 250 mg/kg/day (-10%) and first week at 750 mg/kg/day (-15%). The opposite trend was noted in males given 750 mg/kg/day between weeks 6 and 8 (+8% to +9%; p<0.05). As these differences were of isolated occurrence, not dose-related and/or of opposite trends, they were considered to be of no toxicological importance.No relevant effects were observed on food consumption in the other test item-treated animals during the treatment period.Mean food consumption was slightly higher in previously test item-treated animals during the treatment-free period.See table 3.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No findings were observed at the end of the treatment period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day in males, lower red blood cell parameters (statistically significant for most of them), including mean red blood cell count, mean hemoglobin concentration and mean packed cell volume, were noted. Minimal increase in reticulocyte count was also noted and correlated to the slight decrease in red blood cell mass. However, these differences were mainly due to the contribution of one male (showing blood in urine and renal tubular vacuolation). These effects were considered to be test item treatment-related but of minor toxicological importance as values remained within or were close to the range of historical control values.At 750 mg/kg/day, lower mean eosinophil counts were observed in males and females, whereas higher neutrophil counts were recorded in some males and females suggesting a stress leukogram for 4/10 males and 2/9 females. Prolonged prothrombin time was recorded in males and to a lesser extent in females. In the absence of any other significant changes and since values remained within or were close to the range of historical control values, these differences were considered to be of minor toxicological importance.A decreasing tendency in hemoglobin concentration and packed cell volume was observed in males given 250 mg/kg/day.The other statistically significant differences between control and test item-treated animals, namely in monocyte counts (females given 50 mg/kg/day) were considered to be of no toxicological importance as they were of low magnitude and/or noted with no dose-relationship.No test item-related effects on the hematology parameters were observed at the end of the treatment-free period.See table 4.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

When compared with mean control values, statistically significant blood biochemistry changes were observed:- lower mean sodium levels (-1% in females at 250 mg/kg/day and in males and females at 750 mg/kg/day),- lower mean chloride level (-2% in males at 750 mg/kg/day), - higher mean inorganic phosphorus level (+8 and +15% at 250 mg/kg/day, and +15 and +12% at 750 mg/kg/day, in males and females, respectively),- lower mean protein and albumin levels (-5% in males at 750 mg/kg/day) along with lower mean creatinine level (-9% in males at 750 mg/kg/day),- higher triglyceride levels (+38 and +58% in females at 250 and 750 mg/kg/day, respectively),- higher aspartate aminotransferase activity in males at 750 mg/kg/day (+23%) and higher alanine aminotransferase activity in males and females at 750 mg/kg/day (+88 and +62%, respectively). Although changes in the markers and electrolytes of the renal function (Na+, Cl-, inorganic phosphorus, proteins and albumin) are not consistent with each other, a relationship to microscopic vaculoation of kidneys and/or to vasopressin vacuoles in the pituitary gland could not be excluded.At 750 mg/kg/day, high aspartate aminotransferase activity (> 100 U/L) in 4/10 males and 3/10 females was also associated with high alanine aminotransferase activity in males (> 75 U/L) and females (> 60 U/L) (as well as in 2/10 females at 50 mg/kg/day). These findings were not associated with histological changes in the liver.All the above described findings were considered to be of minor toxicological importance were of minimal magnitude.The other statistically significant differences observed between control and test item-treated animals, namely in the potassium and triglyceride levels (males at 50 mg/kg/day) and total bilirubin level (males at 250 mg/kg/day and males and females at 750 mg/kg/day with many values below the lower limit of quantification) were considered to be within the range of physiological values or incidental and not test item-related as they were of low magnitude, of opposite trend and/or noted with no dose-relationship. Test item-related effects on the blood biochemistry parameters were no longer observed at the end of the treatment-free period.See table 5.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, significant hematuria was noted in 2/10 males and 2/10 females at the end of the treatment period. In the absence of hyperglycemia, glucosuria observed in 3 males could be indicative of a tubular resorption problem. Moreover, in one male, the presence of proteins (= 3 g/L), bilirubin (high-level), nitrites, urobilinogen (= 66 µmol/L) and marked turbid appearance of urine were noted. Higher mean urine volume in males given 250 mg/kg/day, was considered to be incidental and not test item-related as it was of low magnitude and not dose-related.At the end of the treatment-free period, the lower mean urine volumes in males and females previously treated with the high dose level were not considered to be treatment-related as they were not observed at the end of the treatment period and were not associated with any other changes in the urine parameters.See table 6.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

At 750 mg/kg/day, higher, test item-related, mean number of horizontal movements and rearing was noted in males (+24% and +31% vs. controls, respectively) and females (+22% and +76% vs. controls, respectively). There were no correlating clinical signs during the study. This finding observed with a low incidence and mainly due to the contribution of one male and one female was therefore considered to be of minor toxicological importance.At 50 and 250 mg/kg/day, a higher mean number of rearing movements was noted in males and females (between +20 and +44% vs. controls). In view of the slight severity, and as there was no correlating higher mean number of horizontal movements, these findings, mainly due to the contribution of one male and one female at 50 mg/kg/day, were considered to be of no toxicological importance.Differences from controls in grooming and defecation were noted in isolated animals at all dose-levels. In view of the very slight severity and incidence, and in absence of correlating clinical signs during the study, these findings were considered to be unrelated to the test item treatment. Other changes, including alopecia, ptyalism and abdominal breathing, were observed in one or two animals given 50 or 750 mg/kg/day, correlating with clinical signs already observed. Lower mean landing foot splay values were noted in females given 750 mg/kg/day (85 mm vs. 105 mm in controls). These findings were considered to be incidental (alopecia) or were considered to be of minor toxicological importance as they were of isolated occurrence (ptyalism and abdominal breathing) or did not correlate with any other findings (landing foot splay).See table 7.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, there were higher relative-to-body kidney weights in males and females treated at 750 mg/kg/day when compared with controls (+17%, p<0.01 and +14%, p < 0.05, respectively). These organ weight differences were partially related to a lower terminal body weights, with a statistically significant decrease in males. A relationship of these kidney weights changes to microscopic vacuolation of renal tubules could not be established.There were lower absolute and relative-to-body thymus weights in males and females treated at 750 mg/kg/day (up to -24%; statistical significance not reached). This organ weight difference was considered to be related to test item administration and/or stress and correlated with increased incidence and severity of microscopic lymphoid atrophy.All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related. At the end of the treatment-free period, there were slightly higher absolute and/or relative-to-body kidney weights in males and females previously treated at 750 mg/kg/day (up to +14% in males; absolute weights; p<0.01). This organ weight difference was considered not to be related to previous test item administration in males in view of the low magnitude of these differences, of the absence of microscopic correlates and of the absence of kidney weight differences at the end of the treatment period. In females, the higher relative-to-body kidney weight difference was considered to be related with the lower terminal body weights There were lower absolute and relative-to-body thymus weights in females previously treated at 750 mg/kg/day (up to -56% in absolute weights; statistical significance not reached). This organ weight difference was considered to be related to test item administration and/or stress and correlated in part with the minimal increased severity of microscopic lymphoid atrophy in females previously treated at 750 mg/kg/day when compared with controls.Consequently, there was no reversibility of the difference in thymus weights in females. All other weight differences were minimal, did not correlate with macroscopic or microscopic findings and were considered no to be test item treatment-related. These included the higher uterus weights in females previously treated at 750 mg/kg/day probably related with the estrus cycle.See table 8.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled death
There were no test item-related gross findings in the prematurely sacrificed female.
At the end of the treatment periodThere were no test item-related gross findings with the exception of the 1 cm in diameter tan discoloration of the right kidney from 1/10 males treated at 750 mg/kg/day which correlated with moderate renal tubular vacuolation, and the small thymus seen in 1/9 females treated at 750 mg/kg/day which correlated with marked lymphoid atrophy. This affected female had also a marked hematoma in the cranium (see microscopic section).The few other gross findings were considered to be unrelated to test item administration since they were seen also in control group, were not dose-related and/or were considered to be part of the normal background in the rats of these strain and age kept under laboratory condition.
At the end of the treatment-free period
There were no test item-related gross findings suggesting the reversibility or the previous gross findings.The few gross findings were considered to be unrelated to test item administration since they were seen also in control group and/or were considered to be part of the normal background in the rats of these strain and age kept under laboratory condition.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Unscheduled death
One female treated at 750 mg/kg/day and sacrificed on Day 72 had several microscopic findings:- slight vacuolation of the white matter in the brain (hippocampus, cingulum, cerebral peduncle, cerebellum) and in the choroid plexus,- moderate vacuoles in the pars nervosa of the pituitary gland,- marked vacuoles in the renal tubules,- minimal vacuoles in the spleen and in the GALT.The vacuolation in the white matter from the brain was not seen in the test item-treated animals at the end of the treatment period. The vacuoles in the pituitary gland, kidneys, spleen and GALT were seen also in the test item-treated animals at the end of the treatment period at similar or lower severity. These findings were considered to be related with the test item administration and the vacuoles in the brain with extended distribution, kidneys and pars nervosa were considered to have contributed to the moribundity of this animal.
In addition, the following lesions were noted:- slight to moderate atrophy of the ovaries, uterus and vagina,- minimal pancreas degranulation,- increased severity of the adipose tissue in the bone marrow associated with moderate decreased cellularity of the hematopoietic cells,- slight thymus atrophy.These findings may be related with the test item administration and/or stress. The moribundity of this female was considered to be probably related with the test item administration.

At the end of the treatment period
- Vacuoles in several organs.
Minimal to marked vacuoles were seen in the kidneys (tubules and, to a lesser severity, glomeruli), brain (choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and GALT (Gut Associated Lymphoid Tissue) in males and females treated at 750 mg/kg/day and were related with the test item administration. It is noteworthy that these vacuoles were present in the choroid plexus from only 2/10 females treated at 250 mg/kg/day. These vacuoles were round, of moderate to large size (15-50 µm in diameter) and devoid of any staining except those recorded in the pituitary gland which were pale and eosinophilic. The empty vacuoles in brain, spleen, lymph node and GALT were scattered, multifocal while the vacuoles in the pars nervosa were diffuse. An immunohistochemistry staining of vasopressin was performed in the pituitary gland from one control male, two high-dose males and two high-dose females. In the control, there was a diffuse moderate staining of the pars nervosa (pituitary gland), with a more pronounced staining in the neuronal ends. This staining was seen with a higher severity (marked) in the test item treated males and females, with accentuated staining in the neuronal ends and vacuoles seen with hematoxylin/eosin. This suggested that the vacuoles contained vasopressin and may be consistent with enlarged neuronal ends containing increased amount of vasopressin.The pars nervosa from all animals was not present on the submitted slide. That is why the number of lesions out of the number of present pars nervosa was recorded in individual data.
- Forestomach
Dose-related, minimal to slight, focal or multifocal orthokeratotic hyperkeratosis was seen in males and females treated at 250 and 750 mg/kg/day and was related with the test item administration.
- Thymus
Incidence of minimal lymphoid atrophy was seen in some males and females of all control and treated groups. A slight and a marked atrophy were observed in one male and one female treated at 750 mg/kg/day, respectively. This correlated with the lower weights of thymus of these two animals but was not associated with change in the white blood cells count and cellularity. The thymus effects were most probably related with stress.In addition, there was a minimal increased vacuolation in the cortex of the adrenal gland from one male treated at 750 mg/kg/day and in the liver (macrovacuoles; centrilobular location) from one female treated at 750 mg/kg/day. A relationship to test item administration could not be excluded.
There were no test item-related findings in testes after a detailed examination of these organs.
The few other microscopic findings were not considered to be associated with the test item administration because these findings were consistent with spontaneous and background findings described in the literature or their appearance was similar to changes found in controls.Specifically, in one male treated at 250 mg/kg/day, there was a fibrosarcoma in the skin which correlated with macroscopic 2 x 1.5 cm mass. This tumor is not rare in rats. Since it was seen in a single male from intermediate group, it was considered not to be related to the test item administration. One female treated at 750 mg/kg/day had a fibroadenoma in the mammary gland. This tumor is not rare in this strain and is known to occur in rats as young as 12-week old. This animal had also a decidual reaction (or deciduoma) associated with mucification of the vagina, blood and cell debris in the lumen from vagina and uterus. This condition is not rare in the female rats of these strain and age.A PAS staining of the uterus from this female confirm the diagnosis of decidual reaction in the uterus (PAS positivity for the granules in the large cells which suggested that they contain glycogen).
One female treated at 750 mg/kg/day had a large chronic hematoma in the cranium (probably close to the nasal meatus) which correlated with white mass at gross examination. The cause of this hematoma was not clear. It was associated with moderate lymphoid atrophy in the spleen, in the mesenteric lymph node and with minimal to slight degranulation of the salivary gland and pancreas. These last findings were possibly related with stress subsequent to the hematoma in the skull.

At the end of the treatment-free period
- Vacuoles in several organs
Minimal vacuoles were seen in the kidneys (tubules) of one female and in brain (choroid plexus) of two females previously treated at 750 mg/kg/day and was related with the test item administration. No vacuoles were seen in males.This suggested reversibility of these test item treatment-related findings.
- Forestomach
Minimal orthokeratotic hyperkeratosis was seen in 1/5 males and 1/5 females previously treated at 750 mg/kg/day but also in 1/5 control females. This suggested reversibility of these test item treatment-related finding.
- Thymus
Minimal or slight lymphoid atrophy was seen in males and females of the control and/or previously treated at 750 mg/kg/day. For one female treated at 750 mg/kg/day, the slight atrophy was associated with a low weight of thymus, but it was not the case for the other animals presenting a minimal or slight atrophy. These effects seem therefore of spontaneous occurrence.For all these organs, this suggested reversibility of these test item treatment-related finding.
See tables 9 and 10.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

- Estrous cycle (see table 11): There were no statistically significant test item-related effects on mean estrous cycle length or mean number of cycles. However a trend towards an increase in mean estrous cycle length was observed in females given 250 or 750 mg/kg/day at the end of the treatment period. This was considered to be of no toxicological significance in the absence of statistical significance and of differences in estrous cycles at microscopic examination of the genital tract. Differences from controls were no longer observed at the end of the treatment-free period.

- Seminology (no effects observed)
No test item-related effects were noted on the epididymal sperm motility or morphology.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

Key result

Critical effects observed:

no

Table 1: Clinical signs (surviving animals)

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	250	750	0	50	250	750
Thin appearance	-	-	-	1	-	-	-	1
Hunched posture	-	-	-	-	-	1	-	2
Piloerection	-	-	-	-	-	1	-	1
Bent/tilted head	-	-	-	-	-	-	-	1
Abnormal reddish color . urine . vagina	-	-	-	1	- -	- -	- 1	1 1
Soiled urogenital region	-	-	-	-	-	-	-	1
Loud breathing	-	-	2	1	-	-	-	1
Abdominal breathing	-	-	-	-	-	-	-	1
Dyspnea	-	-	-	-	-	-	-	1
Total affected animals	0/15	0/10	2/10	2/15	0/15	1/10	1/10	5/14
Ptyalism	-	-	1	12	-	-	2	14
Reflux at dosing	-	-	-	2	-	-	-	2
Total affected animals	0/15	0/10	1/10	12/15	0/15	1/10	2/10	14/14

-: no clinical signs.

Table 2: Body weight

 

Sex	Male					Female
Dose-level (mg/kg/day)	0	50	250		750	0	50	250	750
Treatment period
Mean BW gain Days 1/29	+102	+110	+101	+72**		+42	+35	+30**	+32
Mean BW gain Days 29/57	+56	+62	+60	+62		+19	+20	+22	+22
Mean BW gain Days 57/92	+35	+31	+31	+20*		+15	+10	+11	+4
Mean BW gain Days 1/92	+194	+202	+191	+154**		+76	+66	+63	+65
% from controls	-	+4	-2	-21		-	-13	-17	-14
Mean body weight on Day 1	418	428	438*	421		264	264	265	267
Mean body weight on Day 92	612	631	629	575		341	330	328	332
% from controls	-	+3	+3	-6		-	-3	-4	-3
Treatment-free period
Mean BW gain Days 92/133	+33	-	-	+58**		+15	-	-	+6*
Mean BW on Day 133	622	-	-	628		366	-	-	348
% from controls	-	-	-	+1		-	-	-	-5

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

Table 3: Food consumption

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	250	750	0	50	250	750
Treatment period
Days 1 to 29	32.1	33.4	33.9	32.2	22.7	21.4	21.4	21.3
Days 29 to 57	30.8	32.5	32.5	33.1	22.3	21.9	21.3	21.8
Days 57 to 92	30.4	31.5	31.1	31.0	21.4	21.1	20.2	20.5
Days 1 to 92	31.0	32.4	32.4	32.0	22.0	21.5	20.9	21.1
% from controls	-	+5	+5	+3	-	-2	-5	-5
Treatment-free period
Days 92 to 133	28.0	-	-	29.8	19.8	-	-	20.7
% from controls	-	-	-	+6	-	-	-	+5

No statistics.

-: not applicable.

Table 4: Hematology

 

Sex	Male
Dose-level (mg/kg/day)	0	50	250	750
End of treatment period
Red blood cell count (T/L)	9.57 (7.96-10.67)	9.28	9.18	8.40** (8.85)^a
Hemoglobin (g/dL)	16.1 (13.3-17.1)	15.5	15.4*	14.4** (15.1)^a
Packed cell volume (L/L)	0.50 (0.41-0.55)	0.48	0.47*	0.44** (0.45)^a
Reticulocytes (%)	2.45 (1.47-3.54)	2.83	2.95	5.37* (3.08)^a
Reticulocytes (T/L)	0.23	0.25	0.27	0.36 (0.27)^a
Neutrophils (G/L)	2.36 (0.80-5.80)	2.15	2.50	2.73 (2.87)^a
Eosinophils (G/L)	0.20 (0.04-0.36)	0.20	0.18	0.02** (0.02)^a
Prothrombin time (s)	22.0 (18.6-23.8)	22.3	22.6	24.4** (24.7)^a
End of treatment-free period
Red blood cell count (T/L)	9.84	/	/	9.71
Hemoglobin (g/dL)	16.0	/	/	16.2
Packed cell volume (L/L)	0.48	/	/	0.49
Reticulocytes (%)	2.09	/	/	2.17
Reticulocytes (T/L)	0.21	/	/	0.20
Neutrophils (G/L)	3.39	/	/	2.70
Eosinophils (G/L)	0.21	/	/	0.19
Prothrombin time (s)	21.2	/	/	19.8

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

( ): minimum-maximum values from historical control data (HCD).

( )^a: excluding animal E25833 from the mean calculation. No statistics.

 

Sex	Female
Dose-level (mg/kg/day)	0	50	250	750
End of treatment period
Red blood cell count (T/L)	8.60 (7.16-9.51)	8.66	8.63	8.33
Hemoglobin (g/dL)	15.4 (13.2-17.0)	15.5	15.4	15.1
Packed cell volume (L/L)	0.46 (0.38-0.51)	0.46	0.46	0.44
Reticulocytes (%)	2.29 (1.15-2.84)	2.25	2.18	3.07
Reticulocytes (T/L)	0.20	0.20	0.19	0.25
Neutrophils (G/L)	1.54 (0.40-2.92)	1.36	1.67	2.28*
Eosinophils (G/L)	0.15 (0.06-0.25)	0.15	0.10	0.02**
Prothrombin time (s)	22.4 (16.5-23.9)	22.4	22.8	23.8
End of treatment-free period
Red blood cell count (T/L)	8.62	/	/	8.90
Hemoglobin (g/dL)	15.5	/	/	15.8
Packed cell volume (L/L)	0.45	/	/	0.46
Reticulocytes (%)	1.78	/	/	1.55
Reticulocytes (T/L)	0.16	/	/	0.14
Neutrophils (G/L)	1.47	/	/	0.94
Eosinophils (G/L)	0.13	/	/	0.11
Prothrombin time (s)	22.9	/	/	23.9

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

( ): minimum-maximum values from historical control data (HCD).

Table 5: Blood biochemistry

 

Sex	Male
Dose-level (mg/kg/day)	0	50	250	750
End of treatment period
Sodium (mmol/L)	144.0	143.9	143.3	142.7**
Chloride (mmol/L)	105.4	105.4	104.4	103.5**
Inorganic phosphorus (mmol/L)	2.09	2.08	2.25*	2.40**
Creatinine (µmol/L)	38.22	36.59	36.14	34.70*
Proteins (g/L)	64.1	64.5	63.3	61.0*
Albumin (g/L)	37	37	36	35**
Triglyceride (mmol/L)	1.03	1.47*	0.89	1.12
Aspartate aminotransferase (U/L)	87	77	87	107*
Alanine aminotransferase (U/L)	40	36	42	75**
End of treatment-free period
Sodium (mmol/L)	143.7	/	/	143.1
Chloride (mmol/L)	105.7	/	/	105.3
Inorganic phosphorus (mmol/L)	2.02	/	/	2.07
Creatinine (µmol/L)	37.10	/	/	36.05
Proteins (g/L)	66.2	/	/	67.2
Albumin (g/L)	37	/	/	37
Aspartate aminotransferase (U/L)	94	/	/	87
Alanine aminotransferase (U/L)	43	/	/	39

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

 

Sex	Female
Dose-level (mg/kg/day)	0	50	250	750
End of treatment period
Sodium (mmol/L)	143.0	143.1	141.9*	141.2**
Chloride (mmol/L)	105.1	106.1	105.1	103.8
Inorganic phosphorus (mmol/L)	1.78	1.65	2.04**	2.00*
Creatinine (µmol/L)	36.13	38.42	36.21	36.28
Proteins (g/L)	66.4	66.5	64.8	64.1
Albumin (g/L)	38	39	38	38
Triglyceride (mmol/L)	0.48	0.48	0.66*	0.76**
Aspartate aminotransferase (U/L)	80	98	73	82
Alanine aminotransferase (U/L)	37	47	35	60*
End of treatment-free period
Sodium (mmol/L)	142.1	/	/	142.1
Chloride (mmol/L)	105.6	/	/	106.5
Inorganic phosphorus (mmol/L)	1.58	/	/	1.44
Creatinine (µmol/L)	41.65	/	/	38.64
Proteins (g/L)	69.9	/	/	68.5
Albumin (g/L)	41	/	/	41
Aspartate aminotransferase (U/L)	90	/	/	75
Alanine aminotransferase (U/L)	45	/	/	30

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

 

Table 6: Urinalysis

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	250	750	0	50	250	750
End of treatment period
Presence of glucose (approx. 5.5 mmol/L)	0	0	0	3	0	0	0	0
Presence of blood
. low	2	1	0	0	0	0	0	1
. moderate	0	0	0	0	0	1	0	1
. high	0	0	0	2	0	0	0	2
Red blood cells	1	4	2	6	0	0	1	3
. 1 - few in some fields	1	4	2	4	0	0	1	1
. 2 - few in all fields	0	0	0	0	0	0	0	1
. 3 - several in all fields	0	0	0	1	0	0	0	1
. 4 - large number in all fields	0	0	0	1	0	0	0	0
End of treatment-free period
Presence of glucose (approx. 5.5 mmol/L)	0	na	na	0	0	na	na	0
Presence of blood	0	na	na	0	0	na	na	0
Red blood cells	0	na	na	0	0	na	na	0

na: not applicable.

Table 7: Motor activity

 

Sex	Male				Female
Dose-level (mg/kg/day)	0	50	250	750	0	50	250	750
Horizontal movements	373	400	371	461	522	590	548	635
% from controls	-	+7	-1	+24	-	+13	+5	+22
Rearing	108	137	130	142	144	207	206	254
% from controls	-	+27	+20	+31	-	+44	+43	+76

-: not applicable.

Table 8: Organ weights

Main organ weight differences in male groups (express in percentage, when compared to controls)

 

Sex	Male
Group	2	3	4
Dose-level (mg/kg/day)	50	250	750
Number of examined animals	10	10	10
Body weight at sacrifice	+1	0	-9*
- Kidneys
.absolute	+8	+6	+6
.relative-to-body	+6	+6	+17**
- Thymus
.absolute	+4	+7	-16
.relative-to-body	+2	+6	-7

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

The significance concerned the organ weights values and not the percentages.

 

Main organ weight differences in female groups (express in percentage, when compared to controls)
at the end of the treatment period

 

Sex	Female
Group	2	3	4
Dose-level (mg/kg/day)	50	250	750
Number of examined animals	10	10	9
Body weight at sacrifice	-1	-2	-4
- Kidneys
.absolute	0	-1	+8
.relative-to-body	+1	+1	+14*
- Thymus
.absolute	+8	+5	-24
.relative-to-body	+9	+7	-24

Statistically significant from controls: *: p<0.05.

The significance concerned the organ weights values and not the percentages.

Table 9: Microscopic findings at tne end of the treatment period

	Males				Females
Group	1	2	3	4	1	2	3	4
Dose-level (mg/kg/day)	0	50	250	750	0	50	250	750
Number of examined individuals	10	10	10	10	10	10	10	9
Kidney; vacuoles; tubules
Grade 1	1	-	-	2	-	-	-	3
Grade 2	-	-	-	-	-	-	-	2
Grade 3	-	-	-	1	-	-	-	-
Grade 4	-	-	-	-	-	-	-	1
Kidney; vacuoles; glomerulus
Grade 1	-	-	-	4	-	-	-	-
Grade 2	-	-	-	-	-	-	-	1
Brain; vacuoles; choroid plexus
Grade 1	-	-	-	9	-	-	2	5
Grade 2	-	-	-	1	-	-	-	3
Pituitary gland*; vacuoles
Grade 1	0/8	1/7	0/6	6/9	0/10	0/9	0/7	3/6
Grade 2	0/8	0/7	0/6	1/9	0/10	0/9	0/7	1/6
Spleen; vacuoles
Grade 1	-	-	-	7	-	-	-	2
Mesenteric lymph node; vacuoles
Grade 1	-	-	-	3	-	na	na	-
GALT; vacuoles
Grade 1	-	-	-	3	-	-	-	2
Thymus; lymphoid atrophy
Grade 1	2	5	5	4	2	1	2	2
Grade 2	-	-	-	1	-	-	-	-
Grade 4	-	-	-	-	-	-	-	1
Forestomach; hyperkeratosis
Grade 1	-	-	1	5	-	-	3	6
Grade 2	-	-	-	-	-	-	-	1

-: not seen in this group.

na: not applicable.

*out of the number of present pars nervosa.

Table 10: Microscopic findings at the end of the treatment-free period 

	Males				Females
Group	1			4	1			4
Dose-level (mg/kg/day)	0			750	0			750
Number of examined individuals	5			5	5			5
Kidney; vacuoles; tubules
Grade 1	-			-	-			1
Brain; vacuoles; choroid plexus
Grade 1	-			-	-			2
Thymus; lymphoid atrophy
Grade 1	-			2	3			2
Grade 2	1			-	-			1
Forestomach; hyperkeratosis
Grade 1	-			1	1			1

-: not seen in this group.

 

Table 11: Estrous cycle

 

Dose-level (mg/kg/day)	0	50	250	750
Treatment period
Number of cycles	4.2	3.9	3.8	3.2
Cycle length (days)	4.2	4.3	5.1	5.9
Number of females having a mean average cycle of 4-5 days	7	6	5	4
Treatment-free period
Number of cycles	1.0	na	na	1.0
Cycle length (days)	4.0	na	na	4.0
Number of females having a mean average cycle of 4-5 days	2	na	na	1

na: not applicable. 

 

Conclusions:

The toxicity of the test item, Diethylaminopropylamine (as pH-neutralized dose formulations), was evaluated after daily oral administration (gavage) to Sprague-Dawley rats at dose-levels of 50, 250 or 750 mg/kg/day for 13 weeks followed by a 6-week treatment-free period. Under the experimental conditions of the study, clinical signs of poor condition were observed at the dose level of 750 mg/kg/day in four females, which induced the premature sacrifice of one of them more severely affected.No other adverse effects were observed in the study.Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 750 mg/kg/day in males and 250 mg/kg/day in females.

Executive summary:

The potential toxicity of Diethylaminopropylamine (as pH-neutralized dose formulations) was evaluated 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). Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item, Diethylaminopropylamine, for 13 weeks as follows: one group of 15 males and 15 females at the dose-level of 750 mg/kg/day (group 4) and two other groups of 10 males and 10 females at dose-levels of 50 (group 2) or 250 (group 3) mg/kg/day. 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 5 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. The actual test item concentrations in the dose formulations prepared for use in Weeks 1, 4, 8 and 13 were determined using agas chromatography with flame ionization detection analytical method. 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 13. Body weight was recorded pre-test, on the first day of treatment and then once a week. Food consumption was recorded weekly. Ophthalmological examinations were performed on all animals before the beginning of the study and on control and test item-treated animals at the end of the treatment period (Week 13). Hematology, blood biochemistry and urinary investigations were performed at the end of the treatment and treatment-free periods (Weeks 13 and 20). Additional blood samples were collected in Weeks 13 and 20 for possible analysis of thyroid hormones levels. The estrous cycle was determined for all females over 21 or 5 consecutive days at the end of the treatment or treatment-free period, respectively. Seminology investigations (count, motility and morphology) were performed on all males at sacrifice at the end of the treatment period. 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 (including testicular staging) was performed on designated tissues from control and high-dose animals sacrificed at the end of the treatment period and from animals that died prematurely and on all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period. PAS staining in the uterus in one high-dose female and immunohistochemistry for vasopressin in the pituitary gland on one control male and two high-dose males and females were examined. The brain, forestomach, gut-associated lymphoid tissue, kidneys, mesenteric lymph nodes (males only), pituitary gland, spleen and thymus of the low- and intermediate-dose animals (groups 2 and 3) sacrificed at the end of the treatment period and of the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment-free period were also microscopically examined as changes were revealed in these organs at the end of the treatment period.

Actual concentrations of Diethylaminopropylamine in the dose formulations administered to the animals during the study remained within an acceptable range (-6.5% to +9.2%) compared to the nominal concentrations. During the treatment period, three females given 750 mg/kg/day showed clinical signs which were considered to be adverse (i.e.hunched posture, dyspnea, abdominal, loud breathing and/or bent head). Hunched posture and piloerection were transiently noted in one female given 50 mg/kg/day. Ptyalism was observed with a dose-related incidence at 250 and 750 mg/kg/day and reflux at dosing was noted in few males and females given 750 mg/kg/day on one occasion.These signscommonly observed when a test item is administered by gavage, were considered not to be an adverse effect. One female given 750 mg/kg/day more severely affected, showed signs of poor clinical condition and therefore was sacrificed in Week 11. Microscopic findings were noted (i.e. vacuoles in the pars nervosa of the pituitary gland, in the renal tubules, in the white matter from the brain, in the choroid plexus, in the spleen and in the GALT). At Functional Observation Battery examination, slightly higher incidences of horizontal movements and rearing were recorded in males and females given 750 mg/kg/day. Body weight gain was slightly lower in males given 750 mg/kg/day during the first and the third months of the treatment period, leading to a slightly lower body weight on completion of the treatment period (-6% vs. controls). Food consumption was not affected by the test item treatment. No ophthalmology findings were observedat the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count were unaffected by the test item treatment. At hematology investigations, minimally to slightly lower hemoglobin concentration and packed cell volume were noted in males from 250 mg/kg/day as well as lower red blood cell count in males given 750 mg/kg/day. This was accompanied with slight increase in reticulocyte count at 750 mg/kg/day. There were also non-adverse lower eosinophil count and prolonged prothrombin time in males and females treated at 750 mg/kg/day.These variations were considered to be of minor toxicological importance. At blood biochemistry investigations, changes in the markers of the renal function (lower sodium and chloride levels, higher inorganic phosphorus levels and lower protein and/or albumin levels) observed in males and/or females from 250 mg/kg/day could be secondary to the electrolytes imbalance induced by the intake of chloride ions used to neutralize the test item. A minimal increase of higher aspartate aminotransferase and alanine aminotransferase activity was observed in males and females given 750 mg/kg/day. All these changes were considered to be of minor importance. At urinary investigations, hematuria in males and females given 750 mg/kg/day along with glucosuria in males were suggestive of a tubular resorption problem. Reversibility of these laboratory findings was noted at the end of the treatment-free period. At the end of the treatment period, microscopic vacuoles were seen in the kidneys (correlated with tan discoloration), brain (in choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and/or GALT in males and females treated at 750 mg/kg/day and at a lesser extent, at 250 mg/kg/day in isolated females. There was also a non-adverse orthokeratotic hyperkeratosis in the forestomach from males and females treated at 750 mg/kg/day and increased severity and incidence of lymphoid atrophy in the thymus from males and surviving females treated at 750 mg/kg/day which correlated with small thymus and lower weights and may be related in part with stress.  

Under the experimental conditions of the study, clinical signs of poor condition were observed at the dose-level of 750 mg/kg/day in four females, which induced the premature sacrifice of one of them more severely affected. No other adverse effects were observed in the study.

Consequently, the NOAEL (No Observed Adverse Effect Level) was established at 750 mg/kg/day in males and 250 mg/kg/day in females.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

analytical purity: separate certificate not enclosed
Purity test date: 21.08.1995
Lot/batch No.: 8/95
Expiration date of the lot/batch: 21.08.1996

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst Aktiengesellschaft, Kastengrund, SPF breeding colony
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: male: 108 g (mean), female: 114 g (mean)
- Housing: in groups of 5
- Diet: ssniff R/M-H (V 1534) ad libitum, except for the period in which the animals were kept in diuresis cages.
- Water: tap water in plastic bottles ad libitum, except for the period in which the animais were kept in diuresis cages.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The determination of the test substance was performed by spectrophotometrical detection (λ = 274 nm).

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily (7x week)

Dose / conc.:

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

Dose / conc.:

10 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)

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a dose range finding study groups of 3 male and 3 female Wistar rats received 3-Dimethylaminopropylamin at dose levels of 50 and 250 mg/kg body weight per day over a period of 14 days. The animals of the 50 mg/kg body weight group showed no clinical signs. The animals of the of the 250 mg/kg body weight showed sporadically swollen abdomen during the study period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were determined before the start of the study and then twice weekly throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the termination of the study
- Anaesthetic used for blood collection: Yes (intrapenitoneal injection of 50-100 mg Ketamin/kg body weight)
- Animals fasted: No data
- How many animals: all
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leucocyte count, thrombocyte count, differential leucocyte count and red ceII morphology, reticulocyte count*, heinz bodies*, coagulation time. *These parameters were scored in the control group and high dose group only.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the termination of the study
- Animals fasted: No data
- How many animals: all
- Parameters examined: Sodium, potassium, inorganic phosphorus, uric acid, bilirubin total, creatinine, glucose, urea, calcium, chloride, aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALATIGPT), alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), cholesterol, triglycerides, total protein, albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from day 26 to day 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance, color, pH-Value, hemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, specific weight, sediment, volume.

OTHERS: The animals were examined weekly for neurological disturbances, damage to the oral mucosa and impairment of dental growth.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes. The necropsy included examination of the heart, stomach, liver, jejunum, kidneys, colon, adrenals, uterus, spleen, ovanies, testes, epididymides, lungs.

Statistics:

The following parameters were compared statistically with the control group values at the level of significance p = 0.05.
Body weights at the designated measurement times, hematological data, clinical chemistry parameters, urine analysis, absolute organ weights and organ to body weight ratios.

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality occurred in four females of the 250 mg/kg body weight group. The animals were found dead on days 11, 18, 22 and 25 of the study.
One male animal of the high dose group showed irregular respiration as well as respiratory sounds at day 11 and 12 of the study. The following clinical signs were observed in female animals of the high dose group sporadically between day 11 and 24 of the study: decreased spontaneous activity, stilted gait, swollen abdomen, respiratory sounds, gasping and panting. The clinical signs were mainly seen in those females which died intercurrently. Behavior and state of health remained unaffected by the administration of the test compound in all other dose groups.
BODY WEIGHT AND WEIGHT GAIN
Body weight development was not impaired by the administration of the test compound and was comparable in all groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
Absolute and relative food consumption remained unaffected by the administration of the test compound throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE
Likewise, the administration of the test compound did not alter the water consumption.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities were observed

HAEMATOLOGY
Hematological examinations revealed statistically significant decreases in erythrocyte counts as weil as in hemoglobine and hematocrit values in males of the intermediate dose group. Haematocrit values were also decreased in males of the high dose group. Leukocyte counts were statistically significantly decreased in males of the intermediate and females of the low dose group. In all cases there was no dose dependency or the values were within the physiological range of rats. Therefore, a compound-related effect is not evident.

CLINICAL CHEMISTRY
Statistical evaluation revealed increases in aspartate aminotransferase and decreases in total protein values in females from the high dose group. lnorganic phosphorus values were decreased in females of the low dose group. Males of the intermediate dose group showed statistically significant decreases in total bilirubin values. In all dose groups of the females the uric acid values were statistically significantly decreased. In all cases there was no dose dependency or the values were within the physiological range of rats. Therefore, a compound-related effect is not evident.

URINALYSIS
Examination of the urine did not reveal any abnormalities.

ORGAN WEIGHTS
In males of the intermediate dose group statistical evaluation of the organ weights revealed increases in relative liver weights. As there was no dose dependency a compound-related effect is not evident.

GROSS PATHOLOGY
In terminally killed animals, no macroscopically visible organ alterations attributable to the compound administration were observed. Dilatation of renal pelvices was found in same male or female animals of all treatment groups and therefore was not considered to be compound-related but to be a strain specific alteration. In the four high dose females which died intercurrently gross findings included discoloration of Iungs with multiple red spots an its surfaces and foamy content. One female also showed a small spleen.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examinations revealed lesions in the four females of the high dose group dying intercurrently. These lesions included congestion of organs, pulmanary hemorrhage, and edema, consistent with cardiorespiratory failure as cause of death. In addition, one of these females exhibited marked loss of lymphatic follicles of the spleen with massive marginal zone and periarteriolar lymphoid sheath atrophy, probably reflecting chronic stress due to treatment. In the one high dose male rat which had shown clinical signs, focal ballooning degeneration of the stratum corneum of the squamous epithellum of the forestomach with granulocytic infiltration of the submucosa was found, most likely due to the locally irritating effect of the compound.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects were seen.

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 3-Dimethylaminopropylamin caused clinical symptoms and mortality in male and female Wistar rats when administered 28 times at the dose level of 250 mg/kg body weight. The female rats seem to be more sensitive.

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Please refer to section 13 for WoE/read-across justification.

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

OECD 407 (DMAPA)

A 28-day oral toxicity study according to OECD guideline 407 was performed on Wistar rats exposed to the test substance at 0, 10, 50, and 250 mg/kg bw/day. One of five high dose males showed impaired respiration. Four out of ten high dose females died. Decreased spontaneous activity, stilted gait, swollen abdomen, and impaired respiration were observed between days 11 and 24, mainly in the females that died. In the four high-dose females that died, macroscopically discoloration of lungs with multiple red spots on its surfaces and foamy content were observed among other findings. Histopathology revealed lesions, which included congestion of organs, pulmonary hemorrhage, and edema. In addition, one of the females exhibited marked loss of lymphatic follicles of the spleen with massive marginal zone and periarteriolar lymphoid sheath atrophy. The high-dose male rat that exhibited clinical signs had focal ballooning degeneration of the squamous epithelium of the fore-stomach. These data demonstrate that DMAPA induces clinical symptoms, systemic toxicity in male and additionally mortality in female rats when administered 28 times during 29 days at the dose level of 250 mg/kg body weight. The female rats seem to be more sensitive. Although a direct systemic-toxic effect on the respiratory tract cannot be excluded, it can be considered as more likely that the observed findings are related to the oral treatment with this corrosive substance. Due to gavage either local placement in the laryngeal/pharyngeal area or secondary aspiration after reflux from the stomach/esophagus appeared reasonable as noted for other corrosive substances too. The NOAEL was 50 mg/kg bw/day (Hoechst 1996; reliability score: 1).

 

OECD 408 (DEAPA)

In a standard subchronic toxicity study (OECD TG 408/GLP) with the structurally related DEAPA Sprague-Dawley rats were treated daily by gavage with the test item for 13 weeks. Male and female rats received dietary target doses of 0, 50, 250 or 750 mg/kg bw/d of DEAPA. During the treatment period, three females given 750 mg/kg bw/d showed clinical signs which were considered to be adverse (i.e. hunched posture, dyspnea, abdominal, loud breathing and/or bent head). One female given 750 mg/kg bw/d more severely affected, showed signs of very poor clinical condition and was sacrificed in week 11. Body weight gain was slightly lower but not adversely effected in males given 750 mg/kg bw/d. Food consumption was not affected by the test item treatment. No ophthalmology findings were observed at the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count were unaffected by the test item treatment. At hematology only minimal to very slight alterations were noted what were finally considered as not adverse. The same was true for none adverse findings in clinical chemistry and urinalysis. Moreover, all changes in hematology, clinical chemistry and urinalysis were completely reversible. At the end of the treatment period, only minor and non-adverse findings were noted in isolated organs and/or animals by histopathology at 750 mg/kg bw/d in both sexes and at 250 mg/kg bw/d in single females. Especially in the females, some of them may be considered as stress-related and due to the irritant potential. Finally, the NOAEL for males was considered to be 750 mg/kg bw/d, and for females 250 mg/kg bw/d predominantly due to the severe clinical findings including death (Consortium Alkylamines 42760 TCR).

 

Conclusion:

The subacute and subchronic studies with DMAPA and DEAPA led to dose related systemic effects in rats but gave no indication of a specific target organ toxicity or accumulation of the substances after repeated dosing.

 

Justification for classification or non-classification

No classification is warranted for repeated dose toxicity according to CLP criteria.