Bis(2-dimethylaminoethyl)(methyl)amine

Endpoint summary

Description of key information

The value of NOAEL (No Observed Adverse Effect Level) for repeated dose toxicity after oral exposure was established as 30 mg/kg bw/day for the male and female. The value was established from the repeated dose 90-day oral toxicity study mainly on the basis of changes in body weight, clinical signs and mortality (one male at the highest dose).
Regarding inhalatory exposure, no systemic effects were revealed, only local irritation, with the local LOAEC of 21.26 mg/m3. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Start of experimental phase: December 16, 2015; Termination of the in-life phase: March 16, 2015

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)

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Servies Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: Males: 55 days, Females: 56 days
- Weight at study initiation: Males: 275.9 g - 310.6 g, Females: 200.7 g - 236.0 g
- Fasting period before study: no
- Housing: singly
- Diet (e.g. ad libitum): conventional laboratory diet ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 7 and 8 days for the male and female main study animals, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C (maximum range)
- Humidity (%): 55% ± 15% (maximum range)
- Air changes (per hr): 15 - 20 times
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: December 16, 2015 (males) or December 17, 2015 (females) To: March 15, 2015 (males) or March 16, 2015 (females)

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item formulations for administration were freshly prepared daily.
The test item was diluted in the tap water to the appropriate concentrations and was
administered orally at a constant administration volume of 2 mL/kg b.w. once daily
for 90 days.

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Concentration in vehicle: 5, 15, 50 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg b.w./day
- Lot/batch no. (if required):
- Purity:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analysed according to the analytical method (Titration with HCl, 1 M) for the determination of the test item in liquid formulation samples validated by LPT.
The measured actual concentrations of the test item-vehicle mixtures were between 98.5% and 103.4% of the nominal concentrations, indicating correctly prepared formulations.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Once daily, 7 days each week for 90 days.

Remarks:

Doses / Concentrations:
10, 30, 100 mg/kg b.w./day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on the results of a 28-day dose-range-finding study (LPT Study No. 32467)
- Rationale for animal assignment (if not random): The rat is a commonly used rodent species for toxicity studies.
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups:
- Section schedule rationale (if not random):

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 a.m. with a final check performed at approximately 4.00 p.m.
- Cage side observations checked in table were included. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and
circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter, detailed clinical observations were made in all animals; in test week 13 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern).
Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, thereafter weekly always on the same day of the week throughout the experimental period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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 AND COMPOUND INTAKE (if drinking water study): No
- Time schedule for examinations:

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before first dosing and at the end of the dosing period.
- Dose groups that were examined: all dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of test week 13 (before necropsy)
- Anaesthetic used for blood collection: Yes (Isoflurane)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: haemoglobin content (HGB), the number of erythrocytes (RBC), leucocytes (WBC), reticulocytes (Reti) and platelets (PCT), the haematocrit value (HCT), the relative and absolute differential blood count, the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH) and the mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of test week 13 (before necropsy)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined: bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), triglycerides, urea in blood, calcium, chloride, potassium, sodium, the albumin/globulin ratio, alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), and lactate dehydrogenase (LDH), Bile Acids, Albumin, Globulin.

URINALYSIS: Yes
- Time schedule for collection of urine: At the end of test week 13 (before necropsy)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight)
- Parameters examined: volume, pH, specific gravity, nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin,
microscopic examination of deposits (Epithelial cells, Leucocytes, Erythrocytes, Organisms, Further constituents (i.e. sperm, casts), Crystalluria)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In test week 13 (before any blood sampling for laboratory examinations)
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity:

OTHER:

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, in all animals of all dose groups
HISTOPATHOLOGY: Yes, restricted to groups 1 (vehicle control) and group 4 (high dose)

Statistics:

Multiple t-test based on DUNNETT, C. W.: Body weight / Food consumption / Haematology and coagulation / Clinical chemistry / Urinalysis / Relative and absolute organ weights
Exact test of R. A. FISHER: Histology
Student’s t-test: All numerical functional tests

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mortality: 100 mg Pentamethyldiethylenetriamine/kg b.w./day: One (no. 64) of 10 male high dose animals died prematurely on test day 90. A reduction in body weight and a severely reduced food intake were noted during the week before death. Clinical signs: Males: No test item-related changes in behaviour or the external appearance were noted for the male rats at any tested dose level during the routine daily observations and the detailed weekly observations. Females: At the high dose level (100 mg Pentamethyldi-ethylenetriamine/kg b.w./day) 2 of 10 animals (nos. 73 and 74) showed piloerection, ptosis, a reduced motility and breathing sounds between test days 7 and 12. As no further changes in behaviour or the external appearance were noted after test day 12 for any animal of the high dose group the observations are considered as test item-related but not as adverse.

Mortality:

mortality observed, treatment-related

Description (incidence):

Mortality: 100 mg Pentamethyldiethylenetriamine/kg b.w./day: One (no. 64) of 10 male high dose animals died prematurely on test day 90. A reduction in body weight and a severely reduced food intake were noted during the week before death. Clinical signs: Males: No test item-related changes in behaviour or the external appearance were noted for the male rats at any tested dose level during the routine daily observations and the detailed weekly observations. Females: At the high dose level (100 mg Pentamethyldi-ethylenetriamine/kg b.w./day) 2 of 10 animals (nos. 73 and 74) showed piloerection, ptosis, a reduced motility and breathing sounds between test days 7 and 12. As no further changes in behaviour or the external appearance were noted after test day 12 for any animal of the high dose group the observations are considered as test item-related but not as adverse.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

100 mg Pentamethyldiethylenetriamine/kg b.w./day: reduced body weight for the males, marginally reduced body weight for the females

Food consumption and compound intake (if feeding study):

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

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):

100 mg Pentamethyldiethylenetriamine/kg b.w./day: a slight reduction in the relative and the absolute kidney weights was noted for the male rats.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Mortality:
Males:
No test item-related premature death was noted for the male animals of the control group and the low and the intermediate dose group (10 or 30 mg test item/kg b.w./day).
At the high dose level (100 mg test item/kg b.w./day) one animal (no. 64) was found dead in the morning on test day 90. A moderate reduction in body weight by 16.2% (from 486.9 g on test day 85 to 408.2 g on test day 90) and a severely reduced food intake were noted for animal no. 64 during the last test week. No other signs of clinical toxicity were noted. Due to autolytic changes, the cause of death could not be determined during the histopathological examination. However, the death is considered as test item-related.
Females:
No test item-related premature death was noted for the female animals of the control group and the treatment groups (10, 30 or 100 mg test item/kg b.w./day).
However, one animal of the high dose group (no. 73) was prematurely sacrificed on test day 9, due to its poor condition (piloerection, ptosis, a reduced motility, breathing sounds and an abdominal position were noted). The histopathological examination revealed a meningitis that was considered as spontaneous and not test item-related.

Clinical signs:Cage-side observations (daily)
Males:
No test item-related changes were noted for the male animals of the low, the intermediate and the high dose group (10, 30 or 100 mg test item/kg b.w./day).
The following observations are considered as spontaneous and not test item-related, as they were only noted for 1 animal each:
Haemorrhagic urine was noted for the male animal no. 30 of the low dose group on 7 consecutive test days from test day 83 until the end of the study on test day 90.
Breathing sounds were noted for the male animal no. 61 of the high dose group on test day 57. The breathing sounds started immediately to 5 min after administration and disappeared between 6 and 24 h after administration.
Females
No test item-related changes were noted for the female animals of the low and in-termediate dose group (10, 30 mg test item/kg b.w./day).
However, at the high dose level (100 mg test item/kg b.w./day) 2 of 10 animals showed piloerection, ptosis, reduced motility and breathing sounds on test days 7 to 9 (no. 73) or 7 to 12 (no. 74).
As the observations were only temporary (no further signs of toxicity were noted for animal no. 74 after test day 12) and no further animals of the high dose group were affected, the observations were considered as test item-related but not adverse.
Beside the above listed observations an abdominal position was additionally noted for animal no. 73 on test day 9, leading to the sacrifice of this animal due to humane reasons on the same day. 'Mortality' the poor condition of animal no. 73 were caused by a not test item-related meningitis. In case of animal no. 73 the other observations (piloerection, ptosis, reduced motility and breathing sounds) could also be related to the observed meningitis.

Detailed clinical observations (weekly)
Males:
No test item-related changes were noted for the male animals of the low, the intermediate and the high dose group (10, 30 or 100 mg test item/kg b.w./day) during the detailed clinical observations, which were performed once weekly outside the home-cage.
The not test-item-related classified observations that were noted for animal no. 30 (haemorrhagic urine) and animal no. 61 (breathing sounds) were confirmed during the process of detailed clinical observations.
Females
No test item-related changes were noted for the female animals of the low, the intermediate and the high dose group (10, 30 or 100 mg test item/kg b.w./day) during the detailed clinical observations.
The afore-mentioned clinical observations in the form of piloerection, ptosis, reduced motility and breathing sounds that were noted for the high dose female animals nos. 73 and 74 were confirmed during the process of detailed clinical observations on test day 8.

BODY WEIGHT AND WEIGHT GAIN
Males:
No test item-related influence on body weight and body weight gain was noted for
the male animals from the low and the intermediate dose group (10 or 30 mg test
item/kg b.w./day).
At the high dose level (100 mg test item/kg b.w./day) a slight but noteworthy reduction
in body weight in comparison to the control group was firstly noted on test
day 57 (6.4% below the value of the control group, not significant). Thereafter,
the difference in body weight between the control group and the high dose group
slightly increased and reached a maximum at the end of the study on test day 90
(9.3% below the value of the control group; p ≤ 0.05). Statistically significant
reductions (at p ≤ 0.05) were noted on test days 78 and 90.
Accordingly, body weight gain from start (test day 1) to the end of the study on
test day 90 was reduced for the rats of the high dose group in comparison to the
rats of the control group and the rats of the low and the intermediate dose group
(see below):

Group / Dose level Body weight gain from test day 1 to 90 (males)
Group 1 (Control) + 80.4%
Group 2 (10 mg/kg) + 78.5%
Group 3 (30 mg/kg) + 73.5%
Group 4 (100 mg/kg) + 64.5% (Group 4 (100 mg/kg) = test item-related change)

Body weight at autopsy for the male rats of the high dose group was also accordingly
reduced (453.3 g in comparison to 486.1 g for the rats of the control group).

Females:
No test item-related differences in body weight and body weight gain were noted between the female animals of the control group and the low and intermediate dose groups (10 or 30 mg test item/kg b.w./day).
A marginal and statistically not significant reduction in body weight was noted for the female animals of the high dose group (100 mg test item/kg b.w./day) after the start of dosing (approx. 3% below the value of the control group; mostly due to animal no. 74 which also showed changes in behaviour and appearance on several test days between the first and second test week.
The marginal reduction in body weight that was noted for the female animals of the high dose group after the start of dosing showed no noteworthy effect on the value of body weight gain for the whole study period (see below).

Group / Dose level Body weight gain from test day 1 to 90 (females)
Group 2 (10 mg/kg) + 44.3%
Group 2 (10 mg/kg) + 42.7%
Group 3 (30 mg/kg) + 38.7%
Group 4 (100 mg/kg) + 39.0%


Body weight at autopsy also revealed no noteworthy differences between the fe-male animals of the control group and the treatment groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)

FOOD EFFICIENCY

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)

OPHTHALMOSCOPIC EXAMINATION

HAEMATOLOGY

CLINICAL CHEMISTRY

URINALYSIS

NEUROBEHAVIOUR

ORGAN WEIGHTS: No test item-related differences were noted between the relative and the absolute organ weights of the male and female animals of the control group and the male and female animals of the low and intermediate dose groups (10 or 30 mg test item/kg b.w./day).A slight reduction in the relative and absolute weight of the kidneys were noted for the male animals of the high dose group (100 mg test item/kg b.w./day) (statistically significant for the right kidney). As no reduction of the kidney weights was noted for the female animals, the reduction noted for the kidney weights of the male rats is considered to be a secondary effect of the reduced body weight of the male rats of the high dose group.
Group /
Dose level Kidney weights of male animals
(% change in comparison to control)
Relative Absolute
left right left right
Group 2
(10 mg/kg) - 3.2 - 4.0 - 5.0 - 5.6
Group 3
(30 mg/kg) - 6.3 - 6.7 - 8.3 - 8.7
Group 4
(100 mg/kg) - 7.7 - 8.2 * - 14.2 - 14.6 *
*/**: p ≤ 0.05/0.01, Dunnett test or Student's t-test.

No test item-related differences between the control group and the treatment groups (10, 30 or 100 mg test item/kg b.w./day) were noted between the relative and the absolute organ weights of the female animals.

GROSS PATHOLOGY

HISTOPATHOLOGY: NON-NEOPLASTIC

HISTOPATHOLOGY: NEOPLASTIC (if applicable)

HISTORICAL CONTROL DATA (if applicable)

OTHER FINDINGS

Dose descriptor:

NOAEL

Effect level:

30 other: mg Pentamethyldiethylenetriamine/kg b.w./day

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

Under the present test conditions the No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg Pentamethyldiethylenetriamine/kg b.w./day for the male and female animals.

Executive summary:

The aim of this study was to obtain information on the toxicity of Pentamethyldiethylenetriamine administered daily by oral administration at doses of 10, 30 or 100 mg/kg b.w./day to CD® rats for 90 test days. At 100 mg Pentamethyldiethylenetriamine/kg b.w./day one of 10 male animals died prematurely on test day 90. No test item-related premature death was noted for the female animals. A reduction in body weight was noted for the male and female animals of the high dose group 100 mg Pentamethyldiethylenetriamine/kg b.w./day. The kidney weights of the male animals of the high dose group (100 mg Pentamethyldiethylenetriamine/ kg b.w./day were reduced. No test item-related effect was noted on the organ weights of the female animals. No test item-related changes in behavior or the external appearance were noted for the male animals. For the female animals changes in the form of piloerection, ptosis, a reduced motility and breathing sounds were noted for 2 of 10 animals. The observations disappeared after a few days and are not considered as adverse. No test item-related influence was noted on the food and drinking water consumption, the haematological and biochemical parameters, the urinary status, the eyes or optic region and the auditory acuity at any of the dose levels. No test item related changes were noted at macroscopic and histopathological inspection. Under the present test conditions the No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg Pentamethyldiethylenetriamine/kg b.w./day for the male and female animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimish score 1 (GLP compliance, no deviations)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, with acceptable restrictions (14-day-exposure only)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: B Charles River Breeding Laboratories, Inc., Kingston, New York.
Acclimating period: 2 weeks prior to exposure
Water and Purina Certified Rodent Chow tf5002 (Riilston Purina Co., St. Louis, Missouri) available ad libitum, except during exposures.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

Rats (5/sex/exposure group)
Exposure: 6 hours/day, 5 days/week.
Number of exposures: 9 exposures in the 0, 3, and 12 ppm groups; 5 exposures in the 48 ppm group.
Chamber airflow maintained at approximately 225 liter/minute.
The air supplied to the chambers controlled by a system designed to maintain temperature at approximately 22°C and relative humidity at approximately 50%.
The temperature and relative humidity in each chamber were recorded approximately hourly during each exposure period.
Vapors of the liquid test material were generated using a J-tube assembly as described by Miller et al. (1980).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration was calculated for each chamber on a daily basis. The analytical chamber concentrations were determined at least 3 times/ exposure period by GC with a flame ionization detector (Varian 1400, Palo Alto, California).
The GC conditions were nitrogen flow = 100 ml/minute, hydrogen flow = 30 ml/minute, and air flow = 300 ml/minute.
A 6' x 1 /8" stainless steel column packed with 10% OV-11 on 100/120 Supelcoport (lot # F16243) was used to separate the test material from air.

Duration of treatment / exposure:

6 hours / day

Frequency of treatment:

5 days / week, 2 weeks

Remarks:

Doses / Concentrations:
0,3,12, or 48 ppm (0,21,85, or 340 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

Duration of exposure - up to 14 days
- Frequency of observations: daily
- Examinations performed: necropsy, histopathology of major organs and tissues, urinalysis, hematology, and clinical chemistry determinations, body weights, major organs weighed.

Positive control:

---

Observations and examinations performed and frequency:

All rats were observed after each exposure period for changes in appearance and overt signs of toxicity.
The observations included evaluation of the eyes, skin, fur, mucous membranes, and respiration. Behavior pattern and nervous system activity was assessed by specific observation for salivation, lacrimation, diarrhea, tremors, convulsions, lethargy, and other signs of central nervous system depression. Records of mortality were maintained. An additional daily observation and observations on weekends
were made. The laboratory veterinarian conducted a pen-light ophthalmological examination on each animal from all exposure groups prior to initiation of exposures and immediately prior to necropsy of animals in the 0,3, and 12 ppm exposure groups. All surviving rats were weighed on
test days 1,3,5,8, and 11 except male and female rats exposed to 48 ppm which were sacrificed on day 8 because of poor clinical condition.

Sacrifice and pathology:

Rats exposed to 0,3, or 12 ppm PMDETA were fasted overnight and sacrificed the day following the last exposure.
Major organs were weighed and tissues evaluated histopathologically. Appropriate samples for urinalysis, hematology, and clinical
chemistry determinations were obtained, body weights recorded, major organs weighed, and tissues were collected for histologic examination.
Nonfasted rats exposed to 48 ppm PMDETA were anesthetized, sacrificed, and necropsied prior to the sixth exposure; blood samples for clinical laboratory determinations, body weights, and organ weights were not obtained.

Other examinations:

Blood samples were obtained by orbital sinus puncture from all surviving rats under slight methoxyflurane anesthesia immediately prior to
necropsy.
Urine was obtained from all rats exposed to 0,3, or 12 ppm PMDETA during the second week of exposure and evaluated for color and
character.

Statistics:

All parameters examined statistically were tested for equality of variances using Bartlett's test. Then, one-way and two-way ANOVA and three-way repeated measures ANOVA were used.
Descriptive statistics (means and SD) were reported for chamber concentrations, temperature, relative humidity, and WBC differential counts.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

irritation of tissues in a direct contact with the test material

Mortality:

mortality observed, treatment-related

Description (incidence):

irritation of tissues in a direct contact with the test material

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Differences were due to decreased mean final body weight in animals.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

irritation of nasal cavity, skin and cornea

Histopathological findings: neoplastic:

no effects observed

Details on results:

ANIMAL OBSERVATIONS:
All rats exposed to 3 ppm survived until termination of the study with no indication of clinical signs as a result of the exposures.
All rats exposed to 12 ppm developed unilateral or bilateral cloudy corneas by test day 5 and these changes persisted throughout
the study.
Following exposure to 48 ppm on test day I, all rats had bilateral cloudy corneas. Most of these rats, after subsequent exposures, developed chromodacryorrhea and swollen, hyperemic ear pinnae with superficial incrustations. Animals from this exposure group were sacrificed
on test day 8 (following 5 exposures) because of their poor physical condition.
BODY WEIGHTS:
The body weights of rats exposed to 3 ppm were comparable to respective control values. An exposure-related time-dose interaction for both
males and females was statistically identified for the 12 and 48 ppm exposure groups. The mean body weights of male rats exposed to 12 ppm were decreased approximately 10% from the corresponding control male value on test day 5 and by test day 11, the difference was approximately 13%.
The mean body weights of male and female rats exposed to 48 ppm were decreased from corresponding control values beginning on test day day 3 and by test day 8, male body weight was decreased approximately 29% from controls while females were decreased approximately 25%.
ORGAN WEIGHTS:
Differences in organ weights were due to decreased mean final body weight in animals.
PATHOLOGY:
Exposure-related gross changes occurred in male and female rats exposed to 12 or 48 ppm while rats exposed to 3 ppm were similar to controls.
Rats exposed to 12 ppm had bilateral cloudy corneas. The 48 pprn exposure group was necropsied on test
day 8 because of their poor physical condition and one female rat from this exposure group was found dead on test day 7. Gross pathologic changes were bilateral cloudy corneas, decreased fat in the abdominal cavity, crusts on the external nares and ear pinnae, and bilateral chromodacryorrhea.
HISTOPATHOLOGY:
Exposure-related and concentration-dependent microscopic changes occurred in the nasal cavity of male and female rats exposed to 3, 12, or 48 ppm, mainly vacuolar degeneration of respiratory epithelium and the underlying submucosal glands. Rats exposed to 12 ppm had vacuolar degeneration of respiratory epithelium and vacuolar degeneration of olfactory epithelium. Additionally, there was vacuolar degeneration of submucosal glands beneath both types of epithelia and the epithelium lining the floor of the anterior portion of the nasopharynx. Several male and female rats in this exposure group had more severe morphologic damage to the nasal cavity as indicated by rnultifocal erosions of respiratory epithelium and inflammatory exudate into the nasal meati. Microscopic changes in the nasal cavities of rats exposed to 48 ppm were vacuolar degeneration of respiratory and submucosal glands throughout all 4 levels of the nasal cavity. There were also extensive areas of erosions, necrosis, and squamous metaplasia of respiratory epithelium. Vacuolar degeneration of nasopharyngeal epithelium extended throughout the nasopharynx.
No additional target organs were identified after microscopic examination of tissues from rats exposed to 3 ppm; however, exposure-related microscopic changes in tissues from rats exposed to 12 or 48 ppm included the eyes, skin, larynx (48 ppm only), trachea (48 ppm only), and bronchi (48 ppm only). Ocular changes were vacuolar degeneration of corneal epithelium.

Dose descriptor:

conc. level: 3 ppm

Effect level:

3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: localised irritation of nasal cavity

Dose descriptor:

conc. level: 12 ppm

Effect level:

12 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local irritation of nasal cavity, skin and cornea

Dose descriptor:

conc. level: 48 ppm

Effect level:

48 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight loss, poor clinical condition, extensive irritation of upper respiratory tract, skin and eyes.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The morphologic appearance of the histopathologic changes was primarily characterized as vacuolar degeneration of epithelium lining the
airways and covering the cornea or skin (superficial layers of the epidermis).
Observed changes were indicative of nonselective localized irritation to tissues at risk by exposure to sufficient
vapor concentrations of the test material. Even at the highest concentration tested (48 ppm), there was no indication of damage to organs or
tissues that were not directly exposed to PMDETA vapors.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2: Comparable to guideline study, with acceptable restrictions (GLP compliance, but 14-day-exposure only)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study, with acceptable restrictions (14-day-exposure only)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

Source: B Charles River Breeding Laboratories, Inc., Kingston, New York.
Acclimating period: 2 weeks prior to exposure
Water and Purina Certified Rodent Chow tf5002 (Riilston Purina Co., St. Louis, Missouri) available ad libitum, except during exposures.

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

Rats (5/sex/exposure group)
Exposure: 6 hours/day, 5 days/week.
Number of exposures: 9 exposures in the 0, 3, and 12 ppm groups; 5 exposures in the 48 ppm group.
Chamber airflow maintained at approximately 225 liter/minute.
The air supplied to the chambers controlled by a system designed to maintain temperature at approximately 22°C and relative humidity at approximately 50%.
The temperature and relative humidity in each chamber were recorded approximately hourly during each exposure period.
Vapors of the liquid test material were generated using a J-tube assembly as described by Miller et al. (1980).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The nominal concentration was calculated for each chamber on a daily basis. The analytical chamber concentrations were determined at least 3 times/ exposure period by GC with a flame ionization detector (Varian 1400, Palo Alto, California).
The GC conditions were nitrogen flow = 100 ml/minute, hydrogen flow = 30 ml/minute, and air flow = 300 ml/minute.
A 6' x 1 /8" stainless steel column packed with 10% OV-11 on 100/120 Supelcoport (lot # F16243) was used to separate the test material from air.

Duration of treatment / exposure:

6 hours / day

Frequency of treatment:

5 days / week, 2 weeks

Remarks:

Doses / Concentrations:
0,3,12, or 48 ppm (0,21,85, or 340 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

Duration of exposure - up to 14 days
- Frequency of observations: daily
- Examinations performed: necropsy, histopathology of major organs and tissues, urinalysis, hematology, and clinical chemistry determinations, body weights, major organs weighed.

Positive control:

---

Observations and examinations performed and frequency:

All rats were observed after each exposure period for changes in appearance and overt signs of toxicity.
The observations included evaluation of the eyes, skin, fur, mucous membranes, and respiration. Behavior pattern and nervous system activity was assessed by specific observation for salivation, lacrimation, diarrhea, tremors, convulsions, lethargy, and other signs of central nervous system depression. Records of mortality were maintained. An additional daily observation and observations on weekends
were made. The laboratory veterinarian conducted a pen-light ophthalmological examination on each animal from all exposure groups prior to initiation of exposures and immediately prior to necropsy of animals in the 0,3, and 12 ppm exposure groups. All surviving rats were weighed on
test days 1,3,5,8, and 11 except male and female rats exposed to 48 ppm which were sacrificed on day 8 because of poor clinical condition.

Sacrifice and pathology:

Rats exposed to 0,3, or 12 ppm PMDETA were fasted overnight and sacrificed the day following the last exposure.
Major organs were weighed and tissues evaluated histopathologically. Appropriate samples for urinalysis, hematology, and clinical
chemistry determinations were obtained, body weights recorded, major organs weighed, and tissues were collected for histologic examination.
Nonfasted rats exposed to 48 ppm PMDETA were anesthetized, sacrificed, and necropsied prior to the sixth exposure; blood samples for clinical laboratory determinations, body weights, and organ weights were not obtained.

Other examinations:

Blood samples were obtained by orbital sinus puncture from all surviving rats under slight methoxyflurane anesthesia immediately prior to
necropsy.
Urine was obtained from all rats exposed to 0,3, or 12 ppm PMDETA during the second week of exposure and evaluated for color and
character.

Statistics:

All parameters examined statistically were tested for equality of variances using Bartlett's test. Then, one-way and two-way ANOVA and three-way repeated measures ANOVA were used.
Descriptive statistics (means and SD) were reported for chamber concentrations, temperature, relative humidity, and WBC differential counts.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

irritation of tissues in a direct contact with the test material

Mortality:

mortality observed, treatment-related

Description (incidence):

irritation of tissues in a direct contact with the test material

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

effects observed, treatment-related

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Differences were due to decreased mean final body weight in animals.

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

irritation of nasal cavity, skin and cornea

Histopathological findings: neoplastic:

no effects observed

Details on results:

ANIMAL OBSERVATIONS:
All rats exposed to 3 ppm survived until termination of the study with no indication of clinical signs as a result of the exposures.
All rats exposed to 12 ppm developed unilateral or bilateral cloudy corneas by test day 5 and these changes persisted throughout
the study.
Following exposure to 48 ppm on test day I, all rats had bilateral cloudy corneas. Most of these rats, after subsequent exposures, developed chromodacryorrhea and swollen, hyperemic ear pinnae with superficial incrustations. Animals from this exposure group were sacrificed
on test day 8 (following 5 exposures) because of their poor physical condition.
BODY WEIGHTS:
The body weights of rats exposed to 3 ppm were comparable to respective control values. An exposure-related time-dose interaction for both
males and females was statistically identified for the 12 and 48 ppm exposure groups. The mean body weights of male rats exposed to 12 ppm were decreased approximately 10% from the corresponding control male value on test day 5 and by test day 11, the difference was approximately 13%.
The mean body weights of male and female rats exposed to 48 ppm were decreased from corresponding control values beginning on test day day 3 and by test day 8, male body weight was decreased approximately 29% from controls while females were decreased approximately 25%.
ORGAN WEIGHTS:
Differences in organ weights were due to decreased mean final body weight in animals.
PATHOLOGY:
Exposure-related gross changes occurred in male and female rats exposed to 12 or 48 ppm while rats exposed to 3 ppm were similar to controls.
Rats exposed to 12 ppm had bilateral cloudy corneas. The 48 pprn exposure group was necropsied on test
day 8 because of their poor physical condition and one female rat from this exposure group was found dead on test day 7. Gross pathologic changes were bilateral cloudy corneas, decreased fat in the abdominal cavity, crusts on the external nares and ear pinnae, and bilateral chromodacryorrhea.
HISTOPATHOLOGY:
Exposure-related and concentration-dependent microscopic changes occurred in the nasal cavity of male and female rats exposed to 3, 12, or 48 ppm, mainly vacuolar degeneration of respiratory epithelium and the underlying submucosal glands. Rats exposed to 12 ppm had vacuolar degeneration of respiratory epithelium and vacuolar degeneration of olfactory epithelium. Additionally, there was vacuolar degeneration of submucosal glands beneath both types of epithelia and the epithelium lining the floor of the anterior portion of the nasopharynx. Several male and female rats in this exposure group had more severe morphologic damage to the nasal cavity as indicated by rnultifocal erosions of respiratory epithelium and inflammatory exudate into the nasal meati. Microscopic changes in the nasal cavities of rats exposed to 48 ppm were vacuolar degeneration of respiratory and submucosal glands throughout all 4 levels of the nasal cavity. There were also extensive areas of erosions, necrosis, and squamous metaplasia of respiratory epithelium. Vacuolar degeneration of nasopharyngeal epithelium extended throughout the nasopharynx.
No additional target organs were identified after microscopic examination of tissues from rats exposed to 3 ppm; however, exposure-related microscopic changes in tissues from rats exposed to 12 or 48 ppm included the eyes, skin, larynx (48 ppm only), trachea (48 ppm only), and bronchi (48 ppm only). Ocular changes were vacuolar degeneration of corneal epithelium.

Dose descriptor:

conc. level: 3 ppm

Effect level:

3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: localised irritation of nasal cavity

Dose descriptor:

conc. level: 12 ppm

Effect level:

12 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: local irritation of nasal cavity, skin and cornea

Dose descriptor:

conc. level: 48 ppm

Effect level:

48 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: body weight loss, poor clinical condition, extensive irritation of upper respiratory tract, skin and eyes.

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

3 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Conclusions:

The morphologic appearance of the histopathologic changes was primarily characterized as vacuolar degeneration of epithelium lining the
airways and covering the cornea or skin (superficial layers of the epidermis).
Observed changes were indicative of nonselective localized irritation to tissues at risk by exposure to sufficient
vapor concentrations of the test material. Even at the highest concentration tested (48 ppm), there was no indication of damage to organs or
tissues that were not directly exposed to PMDETA vapors.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

21.26 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

Klimisch 2: Comparable to guideline study, with acceptable restrictions (GLP compliance, but 14-day-exposure only)

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

For the oral repeated dose toxicity one subacute and one subchronic study is available, for inhalatory exposures one subacute study is available.

The main adverse effects observed in combined repeated dose toxicity study with the reproduction/developmental toxicity screening test after oral exposure (OECD 422) were alteration of the liver function and liver histopathology (vacuolation of hepatocytes in treated males and females at the middle and highest dose level). Vacuolization was also noted in brain, heart, pancreas, lungs, adrenal glands and spleen. However, the results of this study were classified as inconclusive since the satellite groups animals showed reversibility of these adverse effects at terminal necropsy after 14-day recovery period with the exception of the effects on the adrenal glands in high dose females.

 

In the repeated dose 90-day oral toxicity study (OECD 408) one of 10 male animals died prematurely on test day 90 at high dose level (100 mg Pentamethyldiethylenetriamine/kg b.w./day). Due to autolytic changes the cause of death could not be determined, but the death is considered as test item-related. No test item-related premature death was noted for the females.

A reduction in body weight was noted for the male and female animals of the high dose group 100 mg PMDETA/kg bw/day.

The kidney weights of the male animals of the highest dose group (100 mg/kg bw/day) were reduced. No test item-related effect was noted on the organ weights of the female animals.

No adverse effect was observed in behaviour or the external appearance at any tested level. No test item-related influence was noted on the food and drinking water consumption, the haematological and biochemical parameters, the urinary status, the eyes or optic region and the auditory acuity at any of the dose levels. No test item-related changes were noted at macroscopic and histopathological inspection.

Under the present test conditions of the 90-day subchronic repeated dose toxicity after oral exposure the No-Observed-Adverse-Effect-Level (NOAEL) was 30 mg PMDETA/kg bw/day for the male and female animals.

 

Following inhalatory exposure, the main pathological changes were associated with nonselective localized irritation of tissues directly exposed to vapours of the test material (upper respiratory tract, skin and cornea), there was no indication of damage to organs or tissues that were not directly exposed to PMDTA vapors. Pathological changes indicative of localized irritation of tissues exposed to PMDTA vapor occurred at all concentrations, and even at the lowest PMDTA concentration tested (3 ppm = 21.26 mg/m3), all animals were affected (nasal cavity).

 

The lowest concentration tested – 3 ppm was converted to mg/L and to mg/m3, using the following algorithms (OECD TG 39, 2009):

mg/L = ppm x MW / 24,450 = 3 x 173.299 / 24,450 = 0.02126 mg/L

mg/m3 = ppm x MW / 24.45 = 3 x 173.299 / 24.45 = 21.26 mg/m3

MW is molecular weight and 24,450 is a conversion factor at 25 °C.

 

No repeated dose toxicity study via dermal exposure is available.

Justification for classification or non-classification

Based on the available data, the substance is not classified according to CLP Regulation criteria (Regulation (EC) No 1272/2008 on the classification, labelling and packaging of substances and mixtures) for specific target organ toxicity following repeated exposure to PMDETA.

 

The findings from combined repeated dose toxicity study with the reproduction/developmental toxicity screening test were inconclusive and 90-day repeated dose oral toxicity study was conducted.

 

Although the adverse effects noted in 90-day repeated dose oral toxicity study were at the dose level of 100 mg/kg bw day (the guidance value for category 2 is 10 < C ≤ 100), they are considered not to support classification for specific target organ toxicity following repeated exposure. According to CLP Regulation, the small changes in bodyweight or small change in organ weights with no evidence of organ dysfunction do not justify classification.

In this study, only one of ten male animals died prematurely on test day 90. Although due to autolytic changes the cause of death could not be determined, the death was considered as test item-related. No test item-related premature death was noted for the females.

Furthermore, the substance is classified as corrosive and this toxicological effect could reflect just acute toxicity of the substance (i.e. corrosivity).

The results of 90-day repeated dose oral toxicity study did not show any specific target organ toxicity.