Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 2-dimethylamino-2-methyl-1-propanol, DMAMP-80 EUR
- Physical state: clear, colourless liquid
- Analytical purity: 79 ± 0.1% test substance, 19.5 ± 0.12% water
- Impurities (identity and concentrations): 1.316% (uncorrected for water content), unknown impurities
- Purity test date: 18 October 2011
- Lot/batch No.: WI024801S1
- Expiration date of the lot/batch: 24 May 2012 (delivered 25 May 2011) and 31 Aug 2012 (delivered 2 Aug 2011)
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC, USA
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: 161 - 198 g (range, males); 137 - 176 g (range, females)
- Housing: all animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board. Animals were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). Enrichment devices were provided to all animals throughout the study for environmental enrichment and to aid in maintaining the animals’ oral health, and were cleaned weekly.
- Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002 (meal), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.3 - 22.1
- Humidity (%): 41.3 - 56.0
- Air changes (per hr): minimum 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Jun 2011 To: 11 Jan 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% methyl cellulose in deionized water, pH adjusted to 9.0
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance formulations were prepared daily or approximately every 3 days, as necessary, as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored refrigerated. The test substance formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures.


VEHICLE
The vehicle was prepared daily or approximately every 3 days and stored under refrigerated conditions. The pH was adjusted to approximately 9, which is similar to the test substance pH.
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): YD06012N12
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to the initiation of dose administration for study week 0, 2 sets of duplicate samples (exactly 1.0 mL each) for homogeneity and concentration determination were collected from the top, middle, and bottom strata of the 10, 30, and 100 mg/mL dosing formulations and from the middle stratum of the bulk formulation for the control group. In addition, 2 sets of duplicate samples for concentration analysis were collected from the middle stratum of each dosing formulation (including the control group) prepared for dose administration during study weeks 6 and 12. All analyses were conducted using a validated gas chromatography method using flame ionization detection. The actual concentrations were found to be 86.5 - 115% of nominal values, which was within the predefined limits of accuracy and homogeneity (85 - 115%). The RSD was 1.2 - 9.8%, within the 10% limit of acceptability.
Duration of treatment / exposure:
91 - 92 days
Frequency of treatment:
Once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal by gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected by the Sponsor based on the results of a reproductive/developmental screening study (Rasoulpour and Andrus, 2011), in which no systemic effects were observed up to and including the highest dose level of 1000 mg/kg bw/day.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for mortality and moribundity twice daily, and for clinical signs twice daily (at the time of dose administration and 1 - 2 hours after administration)
- Cage side observations included: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and approximately once a week thereafter; and on the last day prior to scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 and approximately weekly thereafter (non-fasted) and on the day of scheduled necropsy (fasted)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption was measured 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: No.


OPHTHALMOSCOPIC EXAMINATION: Yes. The rats were examined using an indirect ophthalmoscope and slit lamp biomicroscope preceded by pupillary dilation with an appropriate mydriatic agent.
- Time schedule for examinations: prior to randomisation and in week 12
- Dose groups that were examined: control group and all treatment groups

HAEMATOLOGY: Yes. Blood was collected from the retro-orbital sinus with the exception of blood for coagulation parameters, which was collected from the vena cava.
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight (at least 16 hours)
- How many animals: control group and treatment groups
- Parameters examined: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, prothrombin time, activated partial thromboplastin time, reticulocyte count (percent), reticulocyte count (absolute), differential leukocyte count (percent and absolute values of: neutrophil, lymphocyte, monocyte, eosinophil, basophil, large unstained cell), red cell distribution width, hemoglobin distribution width, platelet estimate, red cell morphology

CLINICAL CHEMISTRY: Yes. Blood was collected from the retro-orbital sinus.
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes, overnight (at least 16 hours)
- How many animals: control group and treatment groups
- Parameters examined: albumin, total protein, globulin [by calculation], albumin/globulin ratio [by calculation], total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glucose, total cholesterol, calcium, chloride, phosphorus, potassium, sodium, triglycerides, sorbitol dehydrogenase, direct bilirubin, indirect bilirubin

URINALYSIS: Yes
- Time schedule for collection of urine: week 13
- Metabolism cages used for collection of urine: Yes, collected for approximately 16 hrs
- Animals fasted: Yes, overnight
- Parameters examined: specific gravity, pH, urobilinogen, total volume, colour, clarity, protein, glucose, ketones, bilirubin, occult blood, leukocytes, nitrites, microscopy of sediment

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during study week 12, prior to dose administration
- Dose groups that were examined: control group and all treatment groups
- Battery of functions tested: sensory observations (startle response, pupil response, tail pinch response), neuromuscular observations (hind leg and forelimb grip strength), physiological observations (body temperature, body weight), locomotor activity (fine locomotor skill, ambulatory locomotor skills) measured automatically in 60-minute sessions (evaluated over 6 periods: 0 - 10 minutes, 11 - 20 minutes, 21 - 30 minutes, 31 - 40 minutes, 41 - 50 minutes, and 51 - 60 minutes, and as an overall 60-minute test session).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were sacrificed on the day after the final administration (Day 92 or 93). A complete necropsy was conducted on all animals. Animals were euthanized by carbon dioxide inhalation followed by exsanguination. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera.
The absolute and relative weights of the organs listed below were measured for all animals in the control and treatment groups. Paired organs were weighed together and the sum of these weights used. Organs weighed: Brain, thyroids (including parathyroids, weighed after fixation), thymus, heart, liver, spleen, kidneys, adrenals, testes, epididymides, ovaries (without oviducts), uterus with oviducts and pituitary gland.

HISTOPATHOLOGY: Yes
The whole organs and tissues listed below were taken from all the animals, fixed with 10% neutral-buffered formalin, and stored.
Organs fixed: adrenals, aorta, bone with marrow, femur with joint, sternum, bone marrow smear (from femur, not fixed), brain, cerebrum level 1 and level 2, cerebellum with medulla/pons, coagulating glands, cervix, epididymides, eyes with optic nerve, gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), Harderian glands, heart, kidneys, lacrimal glands (exorbital), larynx, liver (3 sections: left and right lobes and right portion of the median lobe), lungs (including bronchi, fixed by inflation with fixative), lymph nodes (axillary, mandibular, mediastinal, mediastinal tissue [adipose and connective tissue around the mediastinal lymph nodes]), mesenteric, mesenteric tissue (adipose and connective tissue attached to the mesenteric lymph nodes), mammary gland (females only), nasal cavity (levels I and III according to the method of Young, 1981), oral tissues (i.e. the palate and teeth [roots of incisors and molars]), ovaries with oviducts (examined if in the plane of section, or if a gross lesion was present), pancreas, peripheral nerve (tibial), pituitary, prostate, salivary glands (mandibular [2]), seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical, thoracic, lumbar), spleen, testes, thymus, thyroid (parathyroids examined if in the plane of section, or if a gross lesion was present), trachea, urinary bladder, uterus, vagina, gross lesions (when possible). Testes and epididymides were fixed in Bouin’s solution, eyes with optic nerves were fixed in Davidson's solution, and then stored in phosphate buffer 10% formalin solution. Axillary and mandibular lymph nodes were not processed to slides.

All listed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with hematoxylin and eosin. Microscopic examination was performed on all tissues processed to slides listed from all animals in the control and 1000 mg/kg bw/day groups at the scheduled necropsy. The kidneys (including the transitional epithelium at the origin of the ureters where they exit the kidneys), liver, lungs, stomach (including the limiting ridge [junction of nonglandular and glandular mucosa]), and gross lesions were examined from all animals in the 100 and 300 mg/kg bw/day groups at the scheduled necropsy. In addition, liver sections were re-prepared and examined for all animals in the control and 1000 mg/kg bw/day groups and sections of the thyroid (with parathyroids) were re-prepared and examined for 2 control group females.

Statistics:
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex. Body weight, body weight change, food consumption, clinical pathology (except gamma glutamyltransferase [GGT]), and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Functional observational battery parameters that yielded scalar or descriptive data were analyzed using Fisher’s Exact Test (Steel and Torrie, 1980). GGT values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. GGT data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: wet, clear matrial around mouth; yellow material in urogenital/anogenital area (non adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: wet, clear matrial around mouth; yellow material in urogenital/anogenital area (non adverse)
Body weight and weight changes:
no effects observed
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:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: changes in lymphocyte levels
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased alkaline phosphatase
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: kidney, liver
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day, female: white areas on lung lobes (non adverse)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: kidneys, slight focally extensive hyperplasia of the transitional epithelium (males); liver, very slight hypertrophy with altered tinctorial properties of centrilobular and midzonal hepatocytes (females)
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There was no mortality during the study period. Wet, clear material was observed around the mouth once during the study in 1/10 males and 2/10 females in the high-dose groups. Yellow, dry or wet material was noted mainly in the urogenital or anogenital area and once on the ventral trunk and hind limb of 1/10 females in the low-dose group and 5/10 females in the high-dose group. The observations were made once on Day 7 and 56, respectively, in the low-dose female and 2/10 high-dose females. In 3 females in the high-dose group, the observation was made up to 5 times during the period of Day 30 to Day 85). These observations may be treatment-related but are not considered to be toxicologically relevant.

BODY WEIGHT AND WEIGHT GAIN
No treatment-related effects on body weight were observed. Significant body weight gain measured once or twice during the study period in a treatment group and a single significant body weight loss in week 13 in females of the low-dose group, were considered to be incidental.

FOOD CONSUMPTION
The mean food consumption was significantly increased in the male low-dose group from study week 10 to 11, but is considered to be a incidental effect as no other dose groups were affected.

OPHTHALMOSCOPIC EXAMINATION
No opthalmic lesions that could be related to the treatment were reported. The prevalence and appearance of all findings observed were typical for laboratory rats of this age and strain.

HAEMATOLOGY
The percentage and absolute number of eosinophils was significantly decreased in both males and females of the high-dose group (see Table 1). In the same female group, the relative percentage and absolute number of lymphocytes was also significantly reduced, while the relative percentage of neutrophiles and monocytes was significantly increased. In females of the highest dose group, the white blood cell level was significantly decreased by approximately 29%, compared with the control group. No similar effect was reported in the males. The changes in blood cell levels are considered to be treatment-related. The toxicological relevance of these changes can not be established. The levels of mean corpuscular hemoglobin and hemoglobin were significantly increased in females in the high-dose level. As the increases were less than 5% and not observed in males, these are not considered to be treatment-related effects. The significant changes in several parameters in low- or mid-dose groups are considered to be incidental, as no dose-relation effect was seen and generally only one sex was affected.

CLINICAL CHEMISTRY
In both males and females of the highest dose group, the phosphorus level was significantly increased (see Table 2). However, as the elevation was 8% (males) and 16% (females) compared to the control group and the results were within the historical data range (Derelanko M.J., Toxicologist's pocket handbook, CRC Press LLC, 2000. ISBN 0-8493-0009-6), this is not considered to be toxicologically relevant. Similarly, the potassium was significantly increased in the female low-dose group and the female and male high-dose groups, respectively. The increases were within the historical data range and 8-12% compared with the control groups, and are seen as not treatment-related.
The alkaline phosphatase concentration was significantly increased (by 31%) in males in the high-dose group and increased by 29% (non-significant) in females. This indicates an increased load on the hepatic metabolism that is treatment-related. However, it is not clear that it is toxicologically relevant. The significant decrease in alanine transferanse in females in the high-dose group, with a non-significant decrease in males in the same dose group, is considered to be incidental.
The cholesterol level in males in the high-dose group was significantly increased, but the lack of a similar effect in females and the relatively low control group level indicates this is not a treatment-related effect. In the high-dose females, the calcium level was significantly increased, but only by 5%, indicating this is not a treatment-related effect. The triglyceride concentration was also significantly increased in the females given the highest dose, but the lack of dose-related increases and similar effects in males indicates this is not treatment-related.

URINALYSIS
The significant increase in urinary volume in females administered the highest dose is probably not treatment-related, as it was not dose-related and no similar increase was noted in males. Notably, there was no effect on urinary pH, despite the test substance pH of approximately 9.

NEUROBEHAVIOUR
There were no significant differences in sensory observations, neuromuscular observations and physiological observation between the control group and treatment groups. The locomotor activity patterns (total activity counts) were unaffected by the test substance administration. There were no significant changes for the test substance-treated groups when compared to the control group at the study week 12 evaluation. The values obtained from the 6 periods evaluated (0-10 minutes, 11-20 minutes, 21-30 minutes, 31-40 minutes, 41-50 minutes, and 51-60 minutes) and the overall 60-minute test session were comparable to the control values. No remarkable shifts in the pattern of adaptation occurred in any of the treatment groups.

ORGAN WEIGHTS
The absolute liver weight, liver weight relative to body weight and liver weight relative to brain weight was significantly increased in males in the low-dose group and in males and females in the high-dose group (see Table 3). Although the weight increases in the highest dose group were 17-22% compared with the control weights, the consistency indicates a toxicologically relevant effect in the highest dose group. The hepatic histopathological changes in the females support this conclusion. For the low-dose group, the result is not considered to be relevant as no effects were seen in the females and no dose-related increase was observed. The high-dose group males and females had significantly higher relative (to final body weight and/or brain weight) kidney weights. The increased kidney weights corresponded to treatment-related microscopic kidney alterations in the males. This is probably a treatment-related effect, but it is not clear if it is toxicologically relevant. The females in the high-dose group had a significantly higher relative (to final body weight) heart weight. This was considered to be unrelated to treatment because the absolute heart weight and heart weight relative to brain weight were not statistically significant at this dose level, and no similar results were seen in the male administered the highest dose. Significant changes in absolute and relative organ weights (heart, epididymes, adrenal glands) observed only in males in the low-dose group were considered to be incidental.

GROSS PATHOLOGY
In 2/10 females of the high-dose group, pinpoint white areas on all lobes of the lung were noted. These macroscopic lung alterations corresponded with the histopathologic observation of very slight multifocal aggregates of alveolar macrophages in both animals. The lung alterations were interpreted by the authors to be a localized response to irritation caused by inadvertent reflux and aspiration of droplets of the test material during the oral gavage dosing procedure. Therefore, the observations were treatment-related, but not toxicologically relevant as droplets are highly unlikely to be inhaled under any exposure conditions. No other treatment-related effects were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
In 8/10 females in the high-dose group, very slight hypertrophy with altered tinctorial properties (increased cytoplasmatic eosinophilia) of centrilobular and midzonal hepatocytes was observed in the liver (see Table 4). In combination with the increase in liver weight, this is considered to be a toxicologically relevant effect. A slight, focally extensive hyperplasia of the transitional epithelium of the kidneys was noted in 5/10 males in the high-dose group and 1/10 females in the control group. The same males in the high-dose group had a very slight chronic inflammation of the transitional epithelium, which was also noted in 1/10 females and 1/10 males in the control group. In 1/10 males in the high-dose group, slight focally extensive unilateral necrosis of the transitional epithelium of the renal papilla was also noted. The kidney effects are considered to be treatment-related, and are probably toxicologically relevant, although only males were affected, as a chronic inflammation is most likely irreversible. Very slight hyperplasia with inflammation of the limiting ridge was observed in 0, 0, 1 and 7/10 males, and in 1, 1, 1, and 4/10 females in the control, low-, mid- and high-dose group, respectively. Slight hyperplasia with inflammation of the limiting ridge in the forestomach and a focal ulcer of the limiting ridge was seen in 1/10 females administered the highest dose. The stomach effects were considered to be the result of direct irritation to the gastric mucosa by the test substance when administered at the highest dose. As humans do not have a forestomach the effect is not relevant to human exposure. Very slight multifocal aggregates of alveolar macrophages was noted in 2/10 females in the high-dose group, which was interpreted by the authors to be a localized response to irritation caused by inadvertent reflux and aspiration of droplets of the test material during the gavage dosing. The change is not considered to be toxicologically relevant. No other treatment-related changes were reported.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical chemistry (alkaline phosphate); organ weights (kidney, liver); histopathology (kidney, liver)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Haematology results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

White blood cells, thous/µL (mean ± SD)

9.01 ± 1.667

10.71 ± 2.38

9.81 ± 2.14

7.99 ± 0.80

6.54 ± 1.65

6.80 ± 1.89

6.31 ± 1.51

4.64 ± 1.45*

Lymphocytes, % of white blood cells

80.0 ± 6.09

81.8 ± 8.53

84.7 ± 4.72

80.3 ± 6.67

83.7 ± 13.92

82.7 ± 3.67

83.3 ± 2.32

77.3 ± 5.63**

Lymphocytes, thous/µL (mean ± SD)

7.28 ± 1.82

8.62 ± 1.13

8.34 ± 1.94

6.44 ± 0.99

5.48 ± 1.49

5.64 ± 1.61

5.27 ± 1.36

3.62 ± 1.31*

Eosinophils, % of white blood cells (mean ± SD)

1.8 ± 1.27

1.4 ± 0.67

1.5 ± 0.42

0.3 ± 0.43**

1.8 ± 0.77

1.7 ± 0.54

1.3 ± 0.39

0.3 ± 0.32**

Eosinophils, thous/µL (mean ± SD)

0.15 ± 0.083

0.14 ± 0.069

0.15 ± 0.062

0.02 ± 0.034**

0.12 ± 0.038

0.11 ± 0.039

0.08 ± 0.037

0.01 ± 0.014**

Neutrophiles, % of white blood cells (mean ± SD)

13.9 ± 4.57

13.0 ± 8.64

10.2 ± 3.75

15.2 ± 9.4

11.5 ± 3.54

12.2 ± 3.34

12.3 ± 2.60

17.0 ± 5.08**

Monocytes, % of white blood cells (mean ± SD)

3.7 ± 2.67

3.2 ± 1.14

2.9 ± 1.38

3.5 ± 2.00

2.3 ± 0.55

2.7 ± 0.77

2.4 ± 0.81

4.7 ± 2.32**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 2: Clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Alkaline phosphatase, IU/L (mean ± SD)

77 ± 19.5

77 ± 17.3

80 ± 8.4

101 ± 19.8**

38 ± 10.4

38 ± 13.3

39 ± 10.4

49 ± 14.4

Alanine aminotransferase, IU/L (mean ± SD)

50 ± 9.1

41 ± 7.4

38 ± 5.7

40 ± 5.5

42 ± 9.1

35 ± 8.4

41 ± 10.9

31 ± 6.5*

Phosphorus, mg/dL (mean ± SD)

7.2 ± 0.4

 

7.4 ± 0.26

7.3 ± 0.26

7.8 ± 0.5**

6.2 ± 0.65

6.4 ± 0.56

6.2 ± 0.60

7.2 ± 0.75**

Potassium, mEq/L (mean ± SD)

4.76 ± 0.41

5.10 ± 0.47

5.11 ± 0.32

5.34 ± 0.37**

4.44 ± 0.40

4.80 ± 0.25*

4.69 ± 0.22

4.80 ± 0.21*

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 3: Organ weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Liver (g)

12.34 ± 1.49

14.45 ± 2.21*

12.90 ± 1.56

14.51 ± 1.60*

7.32 ± 0.72

7.35 ± 0.79

7.59 ± 0.93

8.58 ± 0.88**

Liver (%), relative to body weight

2.59 ± 0.13

2.80 ± 0.19*

2.70 ± 0.20

3.01 ± 0.19**

2.60 ± 0.14

2.68 ± 0.12

2.70 ± 0.12

3.16 ± 0.22**

Liver (%), relative to brain weight

593.27 ± 60.01

707.51± 112.58**

620.61 ± 74.99

701.16 ± 70.36*

379.47 ± 10.56

379.08 ± 34.23

403.86 ± 46.94

446.02 ± 46.97**

Kidneys (g)

3.36 ± 0.37

3.77 ± 0.39

3.48 ± 0.42

3.81 ± 0.46

1.97 ± 0.22

1.93 ± 0.20

1.93 ± 0.22

2.09 ± 0.21

Kidneys (%), relative to body weight

0.708 ± 0.050

0.735 ± 0.044

0.729 ± 0.055

0.792 ± 0.090*

0.70 ± 0.06

0.71 ± 0.03

0.69 ± 0.02

0.77 ± 0.06*

Kidneys (%), relative to brain weight

161.51 ± 13.55

184.30 ± 17.12*

167.40 ± 18.85

184.10 ± 21.91*

102.27 ± 10.90

99.59 ± 8.38

102.70 ± 11.53

109.0 ± 12.12

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 Table 4: Microscopic examination results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Liver, very slight hypertrophy with altered tinctorial properties (increased cytoplasmatic eosinophilia) of centrilobular and midzonal hepatocytes

0/10

0/10

0/10

0/10

0/10

0/10

0/10

8/10

Kidneys, slight focally extensive hyperplasia of the transitional epithelium

0/10

0/10

0/10

5/10

1/10

0/10

0/10

0/10

Kidneys, very slight chronic inflammation of the transitional epithelium

1/10

0/10

0/10

5/10

1/10

0/10

0/10

0/10

Kidney, slight focally extensive unilateral necrosis of the transitional epithelium of the renal papilla

0/10

0/10

0/10

1/10

0/10

0/10

0/10

0/10

Lungs, multifocal aggregates of alveolar macrophages, very slight

2/10

3/10

4/10

3/10

2/10

1/10

1/10

8/10

Stomach, very slight hyperplasia with inflammation of the limiting ridge

0/10

0/10

1/10

7/10

1/10

1/10

1/10

4/10

Stomach, slight hyperplasia with inflammation of the limiting ridge

0/10

0/10

0/10

0/10

0/10

0/10

0/10

1/10

Stomach, focal ulcer of the limiting ridge

0/10

0/10

0/10

0/10

0/10

0/10

0/10

1/10

 

 

 

Applicant's summary and conclusion

Conclusions:
The NOAEL is considered to be 300 mg/kg bw/day, based on the effects observed in the target organs liver (in females) and kidneys (in males),
Executive summary:

A subchronic repeated dose toxicity study was performed according to OECD 408, using 2-(dimethylamino)-2-methylpropan-1-ol (DMAMP) (Wasil, 2012). 10 Crl:CD(SD) rats/sex/dose were administered 0, 100, 300 and 1000 mg/kg bw/day by gavage, for 90 consecutive days.

There was no mortality during the study period. 1/10 females in the low- and 5/10 females in the high-dose group had yellow material on the fur (mainly around the urogenital or anogenital area) up to 5 times during the study period. Although the effect is probably treatment-related, it is not toxicologically relevant. No other treatment-related clinical signs were observed during the study period. There were no treatment-related effects on body weight, body weight gain and food consumption. The opthalmoscopic examination only showed findings that were typical for laboratory rats of the strain and age used. The results of the neurobehavioural tests (functional observation battery) were comparable between the control group and treatment groups. 

In both males and females of the high-dose group, the percentage and absolute number of eosinophils was significantly decreased. In the high-dose female group, the relative percentage and absolute number of lymphocytes was also significantly reduced, while the relative percentage of neutrophiles and monocytes was significantly increased. The white blood cell level in females of the high-dose group was significantly decreased (by approximately 29%), compared with the control group. No similar effect was reported in the males. The observed changes in blood cell levels are considered to be treatment-related; however, the toxicological relevance of these changes could not be established. The alkaline phosphatase concentration was significantly increased (by 31%) in males in the high-dose group and increased by 29% (non-significant) in females. This indicates liver damage that is treatment-related and, considering the hepatic histopathological effects observed in females, toxicologically relevant. In both males and females of the highest dose group, the phosphorus and potassium level was significantly increased. However, as the elevation was approximately 8-16% compared to the control group and the results were within the historical data range (Derelanko M.J., Toxicologist's pocket handbook, CRC Press LLC, 2000. ISBN 0-8493-0009-6), these effects are not considered to be treatment-related.

The absolute liver weight, liver weight relative to body weight and liver weight relative to brain weight was significantly increased in males in the low-dose group and in males and females in the high-dose group. Although the weight increases in the highest dose group were 17-22% compared with the control group, the consistency and the hepatic histopathological changes in the females indicates a toxicologically relevant effect. For the low-dose group, the result is not considered to be relevant as no effects were seen in the females and no dose-related increase was observed. The high-dose group males and females had significantly higher relative (to final body weight and/or brain weight) kidney weights. The weight of the reproductive organs and tissues (ovaries, uterus, epididymides, testes) was comparable between the control and treatment groups. In 2/10 females of the high-dose group pinpoint white areas on all lobes of the lung were noted that corresponded to histopathological lung alterations. The lung alterations were interpreted by the authors to be a localized response to irritation caused by inadvertent reflux and aspiration of droplets of the test material during the oral gavage dosing procedure. Therefore, the observations were treatment-related, but not toxicologically relevant. In 8/10 females in the high-dose group, very slight hypertrophy with altered tinctorial properties (increased cytoplasmatic eosinophilia) of centrilobular and midzonal hepatocytes was observed in the liver. In combination with the increase in liver weight, this is considered to be a toxicologically relevant effect. A slight, focally extensive hyperplasia of the transitional epithelium of the kidneys was noted in 5/10 males in the high-dose group and 1/10 females in the control group. The same males in the high-dose group had a very slight chronic inflammation of the transitional epithelium, which was also noted in 1/10 females and 1/10 males in the control group. In 1/10 males in the high-dose group, slight focally extensive unilateral necrosis of the transitional epithelium of the renal papilla was also noted. Although only males were affected, the kidney effects, together with the increased weight, are considered to be treatment-related.

Very slight hyperplasia with inflammation of the limiting ridge was observed in 7/10 males and 4/10 females in the high-dose group, respectively, and slight hyperplasia was noted in 1/10 females in the same group. 4 cases divided over the remaining groups were probably incidental. The damage to the limiting ridge is most likely caused by local irritation of the test substance (adjusted to pH 9), which was administered in a relatively large volume. As humans do not have a limiting ridge and forestomach, the effect does not have a toxicological relevance to humans. Very slight multifocal aggregates of alveolar macrophages were noted in 2/10 females in the high-dose group, which was interpreted by the authors to be a localized response to irritation caused by inadvertent reflux and aspiration of droplets of the test material during the gavage dosing. There were no treatment-related histopathological findings in the reproductive organs and tissues examined (coagulating glands, cervix, epididymides, ovaries with oviducts prostate, seminal vesicles, testes, uterus, vagina).

Based on the effects observed in the target organs liver (in females) and kidneys (in males), the NOAEL systemic is considered to be 300 mg/kg bw/day.