2-amino-2-methylpropanol

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

11 mg/kg bw/day

Study duration:

chronic

Species:

rat

System:

hepatobiliary

Organ:

liver

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

Study period:

From 02 November 2016 to 07 November 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Principles of method if other than guideline:

- Principle of test: The objective of this study was to determine the toxicity via inhalation exposure of AMP administered by nose-only inhalation during/ after five consecutive daily exposures
- Short description of test conditions: The rats were exposed to three dose levels of test article (700, 1400, and 2000 mg/m3) for six hours per day.
- Parameters analysed / observed:Experimental endpoints consisted of moribundity/mortality and cage-side clinical observations (pre- and post-exposure); body weights; food consumption; clinical pathology parameters (clinical chemistry and hematology); organ weights; and necropsy and histopathological evaluations (lungs, liver, kidney, spleen, adrenals, heart, nasal turbinates, and gross lesions).

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

AMP-REGULAR™ (an aliphatic alkanolamine)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty-two (22) male and 22 female Sprague–Dawley derived rats [Crl:CD®(CD)BR] were obtained from Wilmington, MA-based Charles River Laboratories’ facility in Stone Ridge, NY, and were received at IITRI on October 26, 2016. One day after receipt, weights of animals ranged from 183 to 219 g for males and 152 to 182 g for females. The rats were approximately seven weeks old at arrival (date of birth of September 5, 2016) and approximately eight weeks old at the initiation of dosing.
During the one-week quarantine period, the rats were observed daily for mortality or evidence of moribundity. Before being released from quarantine, the animals were carefully examined (hand-held physical examination) to ensure their health and suitability as test subjects. The rats were released from quarantine on November 2, 2016. Group Assignment and Identification: Animals were randomized with a computerized body weight stratification procedure that produced similar group mean body weight values (not exceeding ±20%) on November 1, 2016, using ToxData®. The animals were randomized into four groups. Each animal selected for the study received a permanent identification number by permanent marker on the tail at the time of randomization. Extra animals not selected for the study were used as training animals. All cages were identified by project number, study number, animal number, dose group, and sex. Cage cards were color-coded by study group.
Food and Water: The rats were provided with Certified Global 18% Protein Rodent Diet [2018C; Teklad Laboratory Animal Diets, Envigo; Madison, WI]. City of Chicago water was supplied by means of an in-cage automatic watering system. No known contaminants that would have interfered with the outcome of the study were present in the food or water of the animals. Reports for the food and water analyses are maintained with facility records. The animals did not have access to food or water during inhalation exposures or to food during the scheduled fasting periods.

Housing and Environment: During quarantine and non-exposure periods of the treatment phase, rats were double-housed in polycarbonate “shoe-box” cages (10 ½” × 19” × 8”) lined with autoclaved absorbent hardwood chip bedding. The cages were equipped with automatic feeding and watering systems. Racks and cages were sanitized following group assignment.
Temperature and relative humidity (%RH) values were recorded once daily during quarantine and twice daily during the treatment period of the study. Recorded values ranged from 21°C to 22°C and from 35% to 51% RH. Fluorescent lighting in the animal room was provided on a cycle of 12 hours of light followed by 12 hours of darkness.
Restraint and Acclimation to Restraint: During the inhalation exposures, the animals were restrained in nose-only holding tubes (CH Technologies, USA; Westwood, NJ). Following confirmation of the correct animal number, each tube was placed in a pre-designated port of the inhalation exposure chamber. Animal placement for each exposure is documented in the study records. Processes for animal tube loading and unloading and tube insertion and removal from the chamber manifold were performed according to laboratory standard operating procedures that are designed to minimize stress to the rats. The rats were observed frequently while restrained to ensure that they remained in the tubes and were not in danger of injury or death. At the end of each exposure, when the chamber was purged of the test substance, the tubes with the animals were removed. The rats were removed from the tubes, observed, and returned to their home cages. The holding tubes were sanitized after each use.
To condition the animals to placement and restraint in nose-only holding tubes and to reduce stress during the exposure phase, the animals were placed in the holding tubes for varying lengths of time on weekdays (but not on any intervening weekend) prior to the start of exposure to the test substance according to the following schedule: 1.5 hours on Day -5; 3 hours on Day -2; and 4.5 hours on Day -1 (October 28 and 31 and November 1, 2016, respectively).
Animal Welfare: The study complied with all applicable sections of the Animal Welfare Act (AWA; Title 9, Code of Federal Regulations), the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (National Institute of Health’s Office of Laboratory Animal Welfare, 2015), and the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). To the extent possible, procedures used in this study were designed to avoid or minimize discomfort, stress, and pain to the animals.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: 10% aqueous solution

Mass median aerodynamic diameter (MMAD):

ca. ca. µm

Remarks on MMAD:

Particle Size Distribution: Mean MMAD values in test atmosphere were 1.10, 0.93, and 1.26 μm for Groups 2-4, respectively. GSD ranges in the test atmosphere were 1.67-1.95, 1.53-1.85, and 1.55-2.07 for Groups 2-4, respectively.

Details on inhalation exposure:

Test Atmosphere Concentration: Overall mean test atmosphere concentrations were 0.697 ± 0.0812, 1.614 ± 0.0986, and 2.004 ± 0.1211 mg/L for Groups 2-4, respectively.
Temperature and Humidity: Overall mean chamber temperatures were 21.8, 21.6, 21.9, and 21.3°C for Groups 1-4, respectively. Overall mean humidity levels were 14.4, 99.9, 99.9, and 99.9% for Groups 1-4, respectively; the elevated relative humidity values for Groups 2-4 were due to the aqueous nature of the dosing formulations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Impinger Analysis: One test atmosphere sample was collected per two hours of exposure (three samples per 6-hour exposure). Chamber aerosol/vapor test atmosphere concentration was monitored with a serially connected train of two impingers, each containing 20 mL of a naphthyl isothiocyanate trapping solution. Samples from each impinger were combined and brought to volume (50 mL) with isopropyl alcohol. These samples were collected at a constant flow rate equal to the port flow of the delivery tube, and the total volume of air sampled was measured with a dry-gas meter. The concentrations of the samples were quantified by chemical analysis at IITRI.
Aerosol Particle Size Distribution: Aerosol particle size distribution was determined once per day for Groups 2-4 with a quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc.; Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the mass accumulated on each collection stage of the QCM.
Aerosol Concentration: Aerosol concentration was monitored with a real-time aerosol sensor (model #pDR-1000AN; MIE Inc., Bedford, MA). The sensor was employed as a real-time indicator of short-term changes in aerosol concentration and was used in guiding laboratory personnel if concentration excursions were encountered.

Duration of treatment / exposure:

Experimental Design: The rats were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days. The first day of exposure was November 2, 2016, and the final day of exposure was November 6, 2016 (Day 5). All study animals were euthanized on November 7, 2016.
Inhalation Exposure Methods
1. Inhalation Exposure Laboratory: The study was conducted in Chambers 1-4 of Laboratory VIII. This laboratory is equipped with 64-port, flow-past-type nose-only inhalation exposure chambers (manufactured by Lab Products Inc.; Seaford, DE). A schematic diagram of the inhalation exposure system is provided in Figure 1.
Air for test atmosphere generation was of breathable quality and was filtered with a compressed air filter and a carbon absorber. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure. During exposure, the animals were held in clear plastic restraining devices (holding tubes; see Section II.C.5) attached to the chamber at the ports.
2. Test Atmosphere Generation: The test atmosphere was generated via aerosolization of the test (10% aqueous solution of AMP-REGULAR™) article using a Pari LC Plus nebulizer (Pari Respiratory Equipment, Inc.; Midlothian, VA). The test article was dispensed into the nebulizer reservoir as needed by means of a syringe. The resulting test atmosphere entered the nose-only inhalation exposure chamber. The exhaust from the exposure chamber was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotameters.

Frequency of treatment:

six hours per day for five days

No. of animals per sex per dose:

5(Male) and 5 (Female) animals per dose group (700, 1400, and 2000 mg/m3)- Total 20 (Male ) and 20 (Female)

Control animals:

yes

yes, concurrent no treatment

Details on study design:

The rats ( 5 male and 5 female per dose group) were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days.

Observations and examinations performed and frequency:

Moribundity/Mortality Observations: Rats were observed for mortality and evidence of moribundity at least once daily prior to the initiation of dosing and at least twice daily during the treatment period. Mortality/moribundity checks were separated by a minimum of four hours, as appropriate.
Physical Examinations/Clinical Observations: Upon initiation of inhalation exposure, animals were observed regularly during exposure to correct any potential emergency conditions while in the holding tubes and to monitor for signs of toxicity (documented manually). Additionally, all surviving study animals were observed at least twice daily (before exposure and within an hour after exposure termination) for clinical signs (documented electronically in ToxData®). Observations included but were not limited to changes in the skin and fur, eyes, and mucous membranes; effects on the respiratory, circulatory, autonomic, and central nervous systems; and effects on somatomotor activity and behavior pattern. Particular attention was devoted to the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Any animals in possibly moribund condition were identified for further monitoring and possible euthanasia.
Body Weights and Body Weight Changes: Animals were weighed one day after receipt; on the day of randomization; and on Study Days 1, 3, and 5. All study animals were fasted overnight and also weighed on Day 6 prior to scheduled necropsy.
Food Consumption: Average food consumption per cage (due to the study animals being double-housed) was recorded on the same schedule as body weights.
Clinical Pathology: Samples were collected for analysis of clinical pathology parameters from all animals prior to scheduled euthanasia on Day 6. Animals were fasted overnight prior to blood collection. Blood samples for hematology and clinical chemistry were obtained from the retro-orbital plexus under anesthesia with 70% CO2/30% O2. The following clinical pathology parameters were evaluated:
a. Hematology: Hematology blood samples were collected into tubes containing EDTA as the anticoagulant. Blood smears were prepared from fresh blood and stained with Wright–Giemsa stain for manual differential leukocyte counts but were to be evaluated only if requested by the Sponsor. The parameters listed below were evaluated using an ADVIA 120 Hematology System Analyzer (Siemens Healthcare Diagnostics; Tarrytown, NY).
Differential white blood cell count (absolute and relative)
Erythrocyte count
Hematocrit
Hemoglobin
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Mean corpuscular volume
Platelet count
Reticulocyte count (absolute and relative)
Total white blood cell count

Clinical Chemistry: Clinical chemistry blood samples were collected into tubes, allowed to clot, centrifuged to obtain serum, and assayed on the day of collection. The parameters listed below were evaluated using a Beckman Coulter AU480 Clinical System (Beckman Coulter, Inc.; Brea, CA).
Alanine aminotransferase
Creatinine
Albumin
Gamma-glutamyl transpeptidase
Albumin/globulin ratio (calculated)
Globulin (calculated)
Alkaline phosphatase
Glucose
Aspartate aminotransferase
Inorganic phosphorus
Bilirubin (total)
Lactate dehydrogenase
Blood urea nitrogen
Potassium
Calcium
Protein (total)
Chloride
Sodium
Cholesterol
Triglycerides

Sacrifice and pathology:

Postmortem Procedures
1. Necropsy, Organ Weights, Tissue Analysis, and Gross Pathology: A complete necropsy was scheduled for all animals on Day 6. Prior to necropsy, the rats were fasted overnight. Animals were euthanized by an overdose of an intraperitoneal injection of sodium pentobarbital and exsanguinated.
At scheduled necropsy, the external surface of the body; all orifices; and the cranial, thoracic, and peritoneal cavities and their contents were examined, and any lesions or abnormal conditions (gross pathologic findings) were recorded. Complete necropsies were performed in the presence/under the supervision of a pathologist.
The tissues listed in the table below were collected and fixed in 10% neutral buffered formalin with the following exceptions: The eyes (with optic nerves) were fixed in Davidson’s solution; the testes and epididymides were fixed in modified
Davidson’s solution; and the bone marrow smear was fixed in methanol. The brain, paired kidneys, liver, adrenals, testes, ovaries, and paired lungs were weighed, and organ-to-body weight ratios were calculated using the fasted body weight for each animal.
Adrenal gland (paired)
Animal Identification (tail)1
Aorta
Bone, femur
Bone, sternum
Bone marrow, femur
Bone marrow, sternum
Bone marrow smear (femur)
Brain
Cervix
Epididymis (paired)
Esophagus
Eye (paired)
Gross lesion (if any)
Harderian gland (paired)
Heart
Kidney (paired)
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Liver
Lung (paired)
Lymph node, mandibular
Lymph node, mesenteric
Mammary gland (females)
Mass (if any)
Nasal cavity and turbinates2
Nerve, optic (paired)
Nerve, sciatic
Ovary (paired)
Pancreas
Parathyroid gland (paired)3
Pituitary gland
Prostate gland
Salivary gland (paired)
Seminal vesicle (paired)
Skeletal muscle
Skin (ventral abdomen)
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord, cervical
Spinal cord, lumbar
Spinal cord, thoracic
Spleen
Stomach
Testis (paired)
Thymus
Thyroid gland (paired)
Trachea
Urinary bladder
Uterus
Vagina
Zymbal gland (paired)3
1 The tail with the identification number of each animal was collected but not processed.
2 Per the request of the Sponsor, as noted in Protocol Amendment No. 2.
3 Due to size constraints, these organs were only evaluated when present in normal sections.
Histopathology: Protocol-specified tissues required for microscopic evaluation (lungs, liver, kidney, spleen, adrenals, heart, and gross lesions) from animals in Groups 1 and 4 (Air Control and High Dose, respectively); liver and gross lesions on the skin (mostly on the nose) from animals in Groups 2 and 3 (Low and Mid Dose, respectively), as noted in Protocol Amendment No. 1; and nasal turbinates from all animals, as noted in Protocol Amendment No. 2 were trimmed, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin by Charles River Laboratories, Pathology Associates, Illinois. Light microscopic evaluation was conducted by a board-certified veterinary pathologist on the protocol-specified tissues.

Statistics:

Statistical Procedures: Descriptive statistics (mean and standard deviation) were calculated, and data were analyzed for statistical significance for body weight/body weight change, clinical pathology (clinical chemistry and hematology), and organ weight/organ-to-body weight ratio data using the ToxData® system. If a data set was normally distributed and of equal variance, statistical comparisons were conducted using a one-way analysis of variance (ANOVA), with post hoc comparisons made (if necessary) using Dunnett’s test. If normality and/or equal variance failed for a data set, statistical comparisons were conducted using nonparametric Kruskal–Wallis ANOVA, with post hoc comparisons made (if necessary) using Dunn’s test. A minimum significance level of p < 0.05 was used for the statistical comparisons in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Post-exposure clinical observations consisted of redness around the eyes and/or nose fur and wet inguinal fur for all animals in all groups, salivation (one Group 1 female, one Group 2 male, two Group 4 males, and four Group 4 females), material around the nose (one Group 2 male), and scab/injury (head/forelimb) in most animals in all three test article-treated groups. Rough coat was also noted in one Group 3 female. Of the above-mentioned signs, those also seen pre-exposure consisted of rough coat (one Group 3 and two Group 4 females); scab/injury (head/forelimb; all Group 3 and 4 males and females and one Group 2 female). The clinical observations noted as scabs were considered test article-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels. Specifically, mean body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid group (Group 3) males and the Mid and High group (Group 3 and 4, respectively) females on Day 5. Mean body weight change was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid and High group males for the Days 1-3, 3-5, and 1-5 intervals. Mean body weight change was also generally decreased for the Mid and High group females, but the changes were not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed.

Food efficiency:

not examined

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

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The effects on % and # MONO a seen in the Mid and/or High dose groups were considered treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The effects on % and # AST (increase) and ALB (decrease) seen in the Mid and/or High dose groups were considered treatment-related.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean fasted body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Group 3 males and the Group 3 and 4 females. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (Group 4 males); liver (Group 3 and 4 males and females); and kidneys (Group 3 and 4 females).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose])
Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose])
Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]),

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, and epidermal hyperplasia) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of squamous epithelium [males], hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).
Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, serocellular crust, and epidermal hyperplasia [males]) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).
Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]), although at lower incidence (one female only) than in the Mid and High groups, and microscopic findings were noted in the skin [females only] (necrosis, ulceration, and serocellular crust) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium [males], squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates [males]).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Remarks on result:

not determinable

Critical effects observed:

yes

Lowest effective dose / conc.:

700 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

All animals survived until scheduled euthanasia. Post-exposure clinical observations consisted of redness around the eyes and/or nose fur and wet inguinal fur for all animals in all groups, salivation (one Group 1 female, one Group 2 male, two Group 4 males, and four Group 4 females), material around the nose (one Group 2 male), and scab/injury (head/forelimb) in most animals in all three test article-treated groups. Rough coat was also noted in one Group 3 female. Of the above-mentioned signs, those also seen pre-exposure consisted of rough coat (one Group 3 and two Group 4 females); scab/injury (head/forelimb; all Group 3 and 4 males and females and one Group 2 female). The clinical observations noted as scabs were considered test article-related.

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels. Specifically, mean body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid group (Group 3) males and the Mid and High group (Group 3 and 4, respectively) females on Day 5. Mean body weight change was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid and High group males for the Days 1-3, 3-5, and 1-5 intervals. Mean body weight change was also generally decreased for the Mid and High group females, but the changes were not statistically significant.

No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed.

The effects on % and # MONO and AST (increase) and ALB (decrease) seen in the Mid and/or High dose groups were considered treatment-related.

Mean fasted body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Group 3 males and the Group 3 and 4 females. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (Group 4 males); liver (Group 3 and 4 males and females); and kidneys (Group 3 and 4 females).

Gross Pathology and Histopathology

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, and epidermal hyperplasia) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of squamous epithelium [males], hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).

Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, serocellular crust, and epidermal hyperplasia [males]) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).

Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]), although at lower incidence (one female only) than in the Mid and High groups, and microscopic findings were noted in the skin [females only] (necrosis, ulceration, and serocellular crust) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium [males], squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates [males]).

Conclusions:

Exposure of rats to AMP-REGULAR™ for 6 hours/day for 5 consecutive days at target concentrations of 700, 1400, and 2000 mg/m3 (Low, Mid, and High, respectively) resulted in no early deaths, with all animals surviving until scheduled euthanasia.
Test article-related clinical observations noted pre- and/or post-exposure consisted of scabbing (head and/or forelimb) in Low (one of five males and one of five females), Mid (five of five males and five of five females), and High (five of five males and five of five females) dose group animals due to irritation (burning) caused by the test article.
In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels, with the change being statistically significant for males. No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed for either sex. In terms of clinical pathology parameters, the effects on absolute and relative monocyte count and aspartate aminotransferase (increase) and albumin (decrease) that were seen in the Mid and/or High dose groups were considered treatment-related. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (High dose males); liver (Mid and High dose males and females); and kidneys (Mid and High dose females).
Adverse histopathological findings seen predominantly in the Mid and High dose animals (and to a lesser extent in the Low dose animals) included observations in the skin (necrosis and ulceration; also noted in the clinical observations) and nasal cavity (atrophy of goblet cells), squamous metaplasia (respiratory and transitional epithelium), and ulceration of the turbinates.
In conclusion, a No-Observed-Adverse-Effect Level (NOAEL) in male and female rats of AMP-REGULAR™ after exposure for 6 hours/day for 5 consecutive days via nose-only inhalation was not determined.

Executive summary:

The objective of this study was to determine the toxicity via inhalation exposure of an aliphatic alkanolamine administered by nose-only inhalation during/after five consecutive daily exposures. The rats were exposed to three dose levels of test article (700, 1400, and 2000 mg/m3) for six hours per day.

Experimental endpoints consisted of moribundity/mortality and cage-side clinical observations (pre- and post-exposure); body weights; food consumption; clinical pathology parameters (clinical chemistry and hematology); organ weights; and necropsy and histopathological evaluations (lungs, liver, kidney, spleen, adrenals, heart, nasal turbinates, and gross lesions).

Overall mean test atmosphere concentrations were 0.697 ± 0.0812, 1.614 ± 0.0986, and 2.004 ± 0.1211 mg/L for Groups 2-4, respectively. Mean MMAD values for the test atmosphere aerosols were 1.10, 0.93, and 1.26 μm for Groups 2-4, respectively. GSD ranges in the test atmosphere were 1.67-1.95, 1.53-1.85, and 1.55-2.07 for Groups 2-4, respectively.

Exposure of rats to AMP-REGULAR™ for 6 hours/day for 5 consecutive days at target concentrations of 700, 1400, and 2000 mg/m3 (Low, Mid, and High, respectively) resulted in no early deaths, with all animals surviving until scheduled euthanasia.

Test article-related clinical observations noted pre- and/or post-exposure consisted of scabbing (head and/or forelimb) in Low (one of five males and one of five females), Mid (five of five males and five of five females), and High (five of five males and five of five females) dose group animals due to irritation (burning) caused by the test article.

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels, with the change being statistically significant for males. No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed for either sex. In terms of clinical pathology parameters,

the effects on absolute and relative monocyte count and aspartate aminotransferase (increase) and albumin (decrease) that were seen in the Mid and/or High dose groups were considered treatment-related. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (High dose males); liver (Mid and High dose males and females); and kidneys (Mid and High dose females).

Adverse histopathological findings seen predominantly in the Mid and High dose animals (and to a lesser extent in the Low dose animals) included observations in the skin (necrosis and ulceration; also noted in the clinical observations) and nasal cavity (atrophy of goblet cells), squamous metaplasia (respiratory and transitional epithelium), and ulceration of the turbinates.

In conclusion, a No-Observed-Adverse-Effect Level (NOAEL) in male and female rats of AMP-REGULAR™ after exposure for 6 hours/day for 5 consecutive days via nose-only inhalation was not determined.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subacute

Species:

rat

System:

hepatobiliary

Organ:

liver

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

Study period:

From 02 November 2016 to 07 November 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Principles of method if other than guideline:

- Principle of test: The objective of this study was to determine the toxicity via inhalation exposure of AMP administered by nose-only inhalation during/ after five consecutive daily exposures
- Short description of test conditions: The rats were exposed to three dose levels of test article (700, 1400, and 2000 mg/m3) for six hours per day.
- Parameters analysed / observed:Experimental endpoints consisted of moribundity/mortality and cage-side clinical observations (pre- and post-exposure); body weights; food consumption; clinical pathology parameters (clinical chemistry and hematology); organ weights; and necropsy and histopathological evaluations (lungs, liver, kidney, spleen, adrenals, heart, nasal turbinates, and gross lesions).

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

AMP-REGULAR™ (an aliphatic alkanolamine)

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Twenty-two (22) male and 22 female Sprague–Dawley derived rats [Crl:CD®(CD)BR] were obtained from Wilmington, MA-based Charles River Laboratories’ facility in Stone Ridge, NY, and were received at IITRI on October 26, 2016. One day after receipt, weights of animals ranged from 183 to 219 g for males and 152 to 182 g for females. The rats were approximately seven weeks old at arrival (date of birth of September 5, 2016) and approximately eight weeks old at the initiation of dosing.
During the one-week quarantine period, the rats were observed daily for mortality or evidence of moribundity. Before being released from quarantine, the animals were carefully examined (hand-held physical examination) to ensure their health and suitability as test subjects. The rats were released from quarantine on November 2, 2016. Group Assignment and Identification: Animals were randomized with a computerized body weight stratification procedure that produced similar group mean body weight values (not exceeding ±20%) on November 1, 2016, using ToxData®. The animals were randomized into four groups. Each animal selected for the study received a permanent identification number by permanent marker on the tail at the time of randomization. Extra animals not selected for the study were used as training animals. All cages were identified by project number, study number, animal number, dose group, and sex. Cage cards were color-coded by study group.
Food and Water: The rats were provided with Certified Global 18% Protein Rodent Diet [2018C; Teklad Laboratory Animal Diets, Envigo; Madison, WI]. City of Chicago water was supplied by means of an in-cage automatic watering system. No known contaminants that would have interfered with the outcome of the study were present in the food or water of the animals. Reports for the food and water analyses are maintained with facility records. The animals did not have access to food or water during inhalation exposures or to food during the scheduled fasting periods.

Housing and Environment: During quarantine and non-exposure periods of the treatment phase, rats were double-housed in polycarbonate “shoe-box” cages (10 ½” × 19” × 8”) lined with autoclaved absorbent hardwood chip bedding. The cages were equipped with automatic feeding and watering systems. Racks and cages were sanitized following group assignment.
Temperature and relative humidity (%RH) values were recorded once daily during quarantine and twice daily during the treatment period of the study. Recorded values ranged from 21°C to 22°C and from 35% to 51% RH. Fluorescent lighting in the animal room was provided on a cycle of 12 hours of light followed by 12 hours of darkness.
Restraint and Acclimation to Restraint: During the inhalation exposures, the animals were restrained in nose-only holding tubes (CH Technologies, USA; Westwood, NJ). Following confirmation of the correct animal number, each tube was placed in a pre-designated port of the inhalation exposure chamber. Animal placement for each exposure is documented in the study records. Processes for animal tube loading and unloading and tube insertion and removal from the chamber manifold were performed according to laboratory standard operating procedures that are designed to minimize stress to the rats. The rats were observed frequently while restrained to ensure that they remained in the tubes and were not in danger of injury or death. At the end of each exposure, when the chamber was purged of the test substance, the tubes with the animals were removed. The rats were removed from the tubes, observed, and returned to their home cages. The holding tubes were sanitized after each use.
To condition the animals to placement and restraint in nose-only holding tubes and to reduce stress during the exposure phase, the animals were placed in the holding tubes for varying lengths of time on weekdays (but not on any intervening weekend) prior to the start of exposure to the test substance according to the following schedule: 1.5 hours on Day -5; 3 hours on Day -2; and 4.5 hours on Day -1 (October 28 and 31 and November 1, 2016, respectively).
Animal Welfare: The study complied with all applicable sections of the Animal Welfare Act (AWA; Title 9, Code of Federal Regulations), the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals (National Institute of Health’s Office of Laboratory Animal Welfare, 2015), and the Guide for the Care and Use of Laboratory Animals (National Research Council, 2011). To the extent possible, procedures used in this study were designed to avoid or minimize discomfort, stress, and pain to the animals.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: 10% aqueous solution

Mass median aerodynamic diameter (MMAD):

ca. ca. µm

Remarks on MMAD:

Particle Size Distribution: Mean MMAD values in test atmosphere were 1.10, 0.93, and 1.26 μm for Groups 2-4, respectively. GSD ranges in the test atmosphere were 1.67-1.95, 1.53-1.85, and 1.55-2.07 for Groups 2-4, respectively.

Details on inhalation exposure:

Test Atmosphere Concentration: Overall mean test atmosphere concentrations were 0.697 ± 0.0812, 1.614 ± 0.0986, and 2.004 ± 0.1211 mg/L for Groups 2-4, respectively.
Temperature and Humidity: Overall mean chamber temperatures were 21.8, 21.6, 21.9, and 21.3°C for Groups 1-4, respectively. Overall mean humidity levels were 14.4, 99.9, 99.9, and 99.9% for Groups 1-4, respectively; the elevated relative humidity values for Groups 2-4 were due to the aqueous nature of the dosing formulations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Impinger Analysis: One test atmosphere sample was collected per two hours of exposure (three samples per 6-hour exposure). Chamber aerosol/vapor test atmosphere concentration was monitored with a serially connected train of two impingers, each containing 20 mL of a naphthyl isothiocyanate trapping solution. Samples from each impinger were combined and brought to volume (50 mL) with isopropyl alcohol. These samples were collected at a constant flow rate equal to the port flow of the delivery tube, and the total volume of air sampled was measured with a dry-gas meter. The concentrations of the samples were quantified by chemical analysis at IITRI.
Aerosol Particle Size Distribution: Aerosol particle size distribution was determined once per day for Groups 2-4 with a quartz crystal microbalance (QCM) cascade impactor (California Measurements Inc.; Sierra Madre, CA) equipped with 10 stages to collect size-segregated samples. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) were calculated from the mass accumulated on each collection stage of the QCM.
Aerosol Concentration: Aerosol concentration was monitored with a real-time aerosol sensor (model #pDR-1000AN; MIE Inc., Bedford, MA). The sensor was employed as a real-time indicator of short-term changes in aerosol concentration and was used in guiding laboratory personnel if concentration excursions were encountered.

Duration of treatment / exposure:

Experimental Design: The rats were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days. The first day of exposure was November 2, 2016, and the final day of exposure was November 6, 2016 (Day 5). All study animals were euthanized on November 7, 2016.
Inhalation Exposure Methods
1. Inhalation Exposure Laboratory: The study was conducted in Chambers 1-4 of Laboratory VIII. This laboratory is equipped with 64-port, flow-past-type nose-only inhalation exposure chambers (manufactured by Lab Products Inc.; Seaford, DE). A schematic diagram of the inhalation exposure system is provided in Figure 1.
Air for test atmosphere generation was of breathable quality and was filtered with a compressed air filter and a carbon absorber. The test atmosphere inlet and exhaust configurations provided a uniform and continuous stream of fresh test atmosphere to the animals undergoing exposure. During exposure, the animals were held in clear plastic restraining devices (holding tubes; see Section II.C.5) attached to the chamber at the ports.
2. Test Atmosphere Generation: The test atmosphere was generated via aerosolization of the test (10% aqueous solution of AMP-REGULAR™) article using a Pari LC Plus nebulizer (Pari Respiratory Equipment, Inc.; Midlothian, VA). The test article was dispensed into the nebulizer reservoir as needed by means of a syringe. The resulting test atmosphere entered the nose-only inhalation exposure chamber. The exhaust from the exposure chamber was moved through a high efficiency particulate air (HEPA) filter by a ring compressor and exhausted outside the building. Inlet and exhaust flows to and from the chamber were controlled and continuously monitored by rotameters.

Frequency of treatment:

six hours per day for five days

No. of animals per sex per dose:

5(Male) and 5 (Female) animals per dose group (700, 1400, and 2000 mg/m3)- Total 20 (Male ) and 20 (Female)

Control animals:

yes

yes, concurrent no treatment

Details on study design:

The rats ( 5 male and 5 female per dose group) were exposed to three levels of test article (700, 1400, and 2000 mg/m3) for six hours per day for five days.

Observations and examinations performed and frequency:

Moribundity/Mortality Observations: Rats were observed for mortality and evidence of moribundity at least once daily prior to the initiation of dosing and at least twice daily during the treatment period. Mortality/moribundity checks were separated by a minimum of four hours, as appropriate.
Physical Examinations/Clinical Observations: Upon initiation of inhalation exposure, animals were observed regularly during exposure to correct any potential emergency conditions while in the holding tubes and to monitor for signs of toxicity (documented manually). Additionally, all surviving study animals were observed at least twice daily (before exposure and within an hour after exposure termination) for clinical signs (documented electronically in ToxData®). Observations included but were not limited to changes in the skin and fur, eyes, and mucous membranes; effects on the respiratory, circulatory, autonomic, and central nervous systems; and effects on somatomotor activity and behavior pattern. Particular attention was devoted to the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep, and coma. Any animals in possibly moribund condition were identified for further monitoring and possible euthanasia.
Body Weights and Body Weight Changes: Animals were weighed one day after receipt; on the day of randomization; and on Study Days 1, 3, and 5. All study animals were fasted overnight and also weighed on Day 6 prior to scheduled necropsy.
Food Consumption: Average food consumption per cage (due to the study animals being double-housed) was recorded on the same schedule as body weights.
Clinical Pathology: Samples were collected for analysis of clinical pathology parameters from all animals prior to scheduled euthanasia on Day 6. Animals were fasted overnight prior to blood collection. Blood samples for hematology and clinical chemistry were obtained from the retro-orbital plexus under anesthesia with 70% CO2/30% O2. The following clinical pathology parameters were evaluated:
a. Hematology: Hematology blood samples were collected into tubes containing EDTA as the anticoagulant. Blood smears were prepared from fresh blood and stained with Wright–Giemsa stain for manual differential leukocyte counts but were to be evaluated only if requested by the Sponsor. The parameters listed below were evaluated using an ADVIA 120 Hematology System Analyzer (Siemens Healthcare Diagnostics; Tarrytown, NY).
Differential white blood cell count (absolute and relative)
Erythrocyte count
Hematocrit
Hemoglobin
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Mean corpuscular volume
Platelet count
Reticulocyte count (absolute and relative)
Total white blood cell count

Clinical Chemistry: Clinical chemistry blood samples were collected into tubes, allowed to clot, centrifuged to obtain serum, and assayed on the day of collection. The parameters listed below were evaluated using a Beckman Coulter AU480 Clinical System (Beckman Coulter, Inc.; Brea, CA).
Alanine aminotransferase
Creatinine
Albumin
Gamma-glutamyl transpeptidase
Albumin/globulin ratio (calculated)
Globulin (calculated)
Alkaline phosphatase
Glucose
Aspartate aminotransferase
Inorganic phosphorus
Bilirubin (total)
Lactate dehydrogenase
Blood urea nitrogen
Potassium
Calcium
Protein (total)
Chloride
Sodium
Cholesterol
Triglycerides

Sacrifice and pathology:

Postmortem Procedures
1. Necropsy, Organ Weights, Tissue Analysis, and Gross Pathology: A complete necropsy was scheduled for all animals on Day 6. Prior to necropsy, the rats were fasted overnight. Animals were euthanized by an overdose of an intraperitoneal injection of sodium pentobarbital and exsanguinated.
At scheduled necropsy, the external surface of the body; all orifices; and the cranial, thoracic, and peritoneal cavities and their contents were examined, and any lesions or abnormal conditions (gross pathologic findings) were recorded. Complete necropsies were performed in the presence/under the supervision of a pathologist.
The tissues listed in the table below were collected and fixed in 10% neutral buffered formalin with the following exceptions: The eyes (with optic nerves) were fixed in Davidson’s solution; the testes and epididymides were fixed in modified
Davidson’s solution; and the bone marrow smear was fixed in methanol. The brain, paired kidneys, liver, adrenals, testes, ovaries, and paired lungs were weighed, and organ-to-body weight ratios were calculated using the fasted body weight for each animal.
Adrenal gland (paired)
Animal Identification (tail)1
Aorta
Bone, femur
Bone, sternum
Bone marrow, femur
Bone marrow, sternum
Bone marrow smear (femur)
Brain
Cervix
Epididymis (paired)
Esophagus
Eye (paired)
Gross lesion (if any)
Harderian gland (paired)
Heart
Kidney (paired)
Large intestine, cecum
Large intestine, colon
Large intestine, rectum
Liver
Lung (paired)
Lymph node, mandibular
Lymph node, mesenteric
Mammary gland (females)
Mass (if any)
Nasal cavity and turbinates2
Nerve, optic (paired)
Nerve, sciatic
Ovary (paired)
Pancreas
Parathyroid gland (paired)3
Pituitary gland
Prostate gland
Salivary gland (paired)
Seminal vesicle (paired)
Skeletal muscle
Skin (ventral abdomen)
Small intestine, duodenum
Small intestine, ileum
Small intestine, jejunum
Spinal cord, cervical
Spinal cord, lumbar
Spinal cord, thoracic
Spleen
Stomach
Testis (paired)
Thymus
Thyroid gland (paired)
Trachea
Urinary bladder
Uterus
Vagina
Zymbal gland (paired)3
1 The tail with the identification number of each animal was collected but not processed.
2 Per the request of the Sponsor, as noted in Protocol Amendment No. 2.
3 Due to size constraints, these organs were only evaluated when present in normal sections.
Histopathology: Protocol-specified tissues required for microscopic evaluation (lungs, liver, kidney, spleen, adrenals, heart, and gross lesions) from animals in Groups 1 and 4 (Air Control and High Dose, respectively); liver and gross lesions on the skin (mostly on the nose) from animals in Groups 2 and 3 (Low and Mid Dose, respectively), as noted in Protocol Amendment No. 1; and nasal turbinates from all animals, as noted in Protocol Amendment No. 2 were trimmed, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin by Charles River Laboratories, Pathology Associates, Illinois. Light microscopic evaluation was conducted by a board-certified veterinary pathologist on the protocol-specified tissues.

Statistics:

Statistical Procedures: Descriptive statistics (mean and standard deviation) were calculated, and data were analyzed for statistical significance for body weight/body weight change, clinical pathology (clinical chemistry and hematology), and organ weight/organ-to-body weight ratio data using the ToxData® system. If a data set was normally distributed and of equal variance, statistical comparisons were conducted using a one-way analysis of variance (ANOVA), with post hoc comparisons made (if necessary) using Dunnett’s test. If normality and/or equal variance failed for a data set, statistical comparisons were conducted using nonparametric Kruskal–Wallis ANOVA, with post hoc comparisons made (if necessary) using Dunn’s test. A minimum significance level of p < 0.05 was used for the statistical comparisons in this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Post-exposure clinical observations consisted of redness around the eyes and/or nose fur and wet inguinal fur for all animals in all groups, salivation (one Group 1 female, one Group 2 male, two Group 4 males, and four Group 4 females), material around the nose (one Group 2 male), and scab/injury (head/forelimb) in most animals in all three test article-treated groups. Rough coat was also noted in one Group 3 female. Of the above-mentioned signs, those also seen pre-exposure consisted of rough coat (one Group 3 and two Group 4 females); scab/injury (head/forelimb; all Group 3 and 4 males and females and one Group 2 female). The clinical observations noted as scabs were considered test article-related.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled euthanasia.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels. Specifically, mean body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid group (Group 3) males and the Mid and High group (Group 3 and 4, respectively) females on Day 5. Mean body weight change was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid and High group males for the Days 1-3, 3-5, and 1-5 intervals. Mean body weight change was also generally decreased for the Mid and High group females, but the changes were not statistically significant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed.

Food efficiency:

not examined

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

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The effects on % and # MONO a seen in the Mid and/or High dose groups were considered treatment-related.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The effects on % and # AST (increase) and ALB (decrease) seen in the Mid and/or High dose groups were considered treatment-related.

Urinalysis findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Mean fasted body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Group 3 males and the Group 3 and 4 females. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (Group 4 males); liver (Group 3 and 4 males and females); and kidneys (Group 3 and 4 females).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose])
Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose])
Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]),

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, and epidermal hyperplasia) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of squamous epithelium [males], hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).
Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, serocellular crust, and epidermal hyperplasia [males]) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).
Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]), although at lower incidence (one female only) than in the Mid and High groups, and microscopic findings were noted in the skin [females only] (necrosis, ulceration, and serocellular crust) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium [males], squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates [males]).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Key result

Remarks on result:

not determinable

Critical effects observed:

yes

Lowest effective dose / conc.:

700 mg/m³ air (nominal)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

All animals survived until scheduled euthanasia. Post-exposure clinical observations consisted of redness around the eyes and/or nose fur and wet inguinal fur for all animals in all groups, salivation (one Group 1 female, one Group 2 male, two Group 4 males, and four Group 4 females), material around the nose (one Group 2 male), and scab/injury (head/forelimb) in most animals in all three test article-treated groups. Rough coat was also noted in one Group 3 female. Of the above-mentioned signs, those also seen pre-exposure consisted of rough coat (one Group 3 and two Group 4 females); scab/injury (head/forelimb; all Group 3 and 4 males and females and one Group 2 female). The clinical observations noted as scabs were considered test article-related.

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels. Specifically, mean body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid group (Group 3) males and the Mid and High group (Group 3 and 4, respectively) females on Day 5. Mean body weight change was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Mid and High group males for the Days 1-3, 3-5, and 1-5 intervals. Mean body weight change was also generally decreased for the Mid and High group females, but the changes were not statistically significant.

No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed.

The effects on % and # MONO and AST (increase) and ALB (decrease) seen in the Mid and/or High dose groups were considered treatment-related.

Mean fasted body weight was statistically significantly decreased in comparison to the Air Control group (Group 1) for the Group 3 males and the Group 3 and 4 females. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (Group 4 males); liver (Group 3 and 4 males and females); and kidneys (Group 3 and 4 females).

Gross Pathology and Histopathology

High Dose (2000 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, and epidermal hyperplasia) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of squamous epithelium [males], hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).

Mid Dose (1400 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]) and microscopic findings were noted in the liver (vacuolation) and skin (necrosis, ulceration, mixed cell infiltrates, serocellular crust, and epidermal hyperplasia [males]) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium, squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates).

Low Dose (700 mg/m3): At necropsy, gross findings were noted in the skin (crust [predominantly on the nose]), although at lower incidence (one female only) than in the Mid and High groups, and microscopic findings were noted in the skin [females only] (necrosis, ulceration, and serocellular crust) and body cavity, nasal (atrophy of goblet and olfactory cells, mixed cell infiltrates, hyperplasia of transitional epithelium [males], squamous metaplasia of respiratory and transitional epithelium, and ulceration of turbinates [males]).

Conclusions:

Exposure of rats to AMP-REGULAR™ for 6 hours/day for 5 consecutive days at target concentrations of 700, 1400, and 2000 mg/m3 (Low, Mid, and High, respectively) resulted in no early deaths, with all animals surviving until scheduled euthanasia.
Test article-related clinical observations noted pre- and/or post-exposure consisted of scabbing (head and/or forelimb) in Low (one of five males and one of five females), Mid (five of five males and five of five females), and High (five of five males and five of five females) dose group animals due to irritation (burning) caused by the test article.
In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels, with the change being statistically significant for males. No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed for either sex. In terms of clinical pathology parameters, the effects on absolute and relative monocyte count and aspartate aminotransferase (increase) and albumin (decrease) that were seen in the Mid and/or High dose groups were considered treatment-related. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (High dose males); liver (Mid and High dose males and females); and kidneys (Mid and High dose females).
Adverse histopathological findings seen predominantly in the Mid and High dose animals (and to a lesser extent in the Low dose animals) included observations in the skin (necrosis and ulceration; also noted in the clinical observations) and nasal cavity (atrophy of goblet cells), squamous metaplasia (respiratory and transitional epithelium), and ulceration of the turbinates.
In conclusion, a No-Observed-Adverse-Effect Level (NOAEL) in male and female rats of AMP-REGULAR™ after exposure for 6 hours/day for 5 consecutive days via nose-only inhalation was not determined.

Executive summary:

The objective of this study was to determine the toxicity via inhalation exposure of an aliphatic alkanolamine administered by nose-only inhalation during/after five consecutive daily exposures. The rats were exposed to three dose levels of test article (700, 1400, and 2000 mg/m3) for six hours per day.

Experimental endpoints consisted of moribundity/mortality and cage-side clinical observations (pre- and post-exposure); body weights; food consumption; clinical pathology parameters (clinical chemistry and hematology); organ weights; and necropsy and histopathological evaluations (lungs, liver, kidney, spleen, adrenals, heart, nasal turbinates, and gross lesions).

Overall mean test atmosphere concentrations were 0.697 ± 0.0812, 1.614 ± 0.0986, and 2.004 ± 0.1211 mg/L for Groups 2-4, respectively. Mean MMAD values for the test atmosphere aerosols were 1.10, 0.93, and 1.26 μm for Groups 2-4, respectively. GSD ranges in the test atmosphere were 1.67-1.95, 1.53-1.85, and 1.55-2.07 for Groups 2-4, respectively.

Exposure of rats to AMP-REGULAR™ for 6 hours/day for 5 consecutive days at target concentrations of 700, 1400, and 2000 mg/m3 (Low, Mid, and High, respectively) resulted in no early deaths, with all animals surviving until scheduled euthanasia.

Test article-related clinical observations noted pre- and/or post-exposure consisted of scabbing (head and/or forelimb) in Low (one of five males and one of five females), Mid (five of five males and five of five females), and High (five of five males and five of five females) dose group animals due to irritation (burning) caused by the test article.

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels, with the change being statistically significant for males. No statistically significant differences in food consumption for the test article-treated groups in comparison to the Air Control group (Group 1) were observed for either sex. In terms of clinical pathology parameters,

the effects on absolute and relative monocyte count and aspartate aminotransferase (increase) and albumin (decrease) that were seen in the Mid and/or High dose groups were considered treatment-related. Statistically significant increases in mean relative organ weight in comparison to the control group were seen in adrenals (High dose males); liver (Mid and High dose males and females); and kidneys (Mid and High dose females).

Adverse histopathological findings seen predominantly in the Mid and High dose animals (and to a lesser extent in the Low dose animals) included observations in the skin (necrosis and ulceration; also noted in the clinical observations) and nasal cavity (atrophy of goblet cells), squamous metaplasia (respiratory and transitional epithelium), and ulceration of the turbinates.

In conclusion, a No-Observed-Adverse-Effect Level (NOAEL) in male and female rats of AMP-REGULAR™ after exposure for 6 hours/day for 5 consecutive days via nose-only inhalation was not determined.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

700 mg/m³

Study duration:

subacute

Species:

rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Species:

rat

Additional information

This is a summary of a more detailed assessment provided in attachment to this section.

Oral route:

Due to its long history of use and vast applications, extensive data are available on the repeated dose oral toxicity of AMP (including via reproduction and developmental toxicity studies), tested under multiple forms (as AMP or as AMP HCl) and regimes (in diet or by gavage), in multiple species (rat, mouse, rabbit, dog, monkey) and over multiple dosing periods (5 day, 4 weeks, 8 weeks, 10 weeks in OECD 443, 3 months and 1 year). In all species, oral dosing was well tolerated, with the exception of an old 5-day study in rats and monkeys where high doses led to significant gastrointestinal irritation and distress due to alkalinity of the test item (pH 11). With the exception of the 8-week study in mice where no significant toxicity was observed, the liver was the target organ for AMP in all species. AMP caused a dose-related accumulation of lipids in hepatocytes (liver steatosis), with an increase in liver weight and in the more severely affected animals also increased blood levels of liver enzymes and hepatocyte vacuolation. Excluding local effects caused by the alkalinity of the test material, no other significant repeated dose toxic effects were noted. There was no clear trend concerning compared sensitivity between males and females. Inter-spieces comparison shows that mice were less sensitive than other species.

In the 28-day dog study, the lowest dose tested (19 mg/kg bw) appeared to be a no observed adverse effect level due to liver toxicity observed in the mid and high dose groups, however even at this dose there was some evidence of a minor (not adverse) liver effect in the female dog (the male dog showed no signs of effects).

In the 3-month dog study similar effects were observed. There were minimal effects in the dogs dosed with approximately 15 mg/kg bw/day, but one female dog dosed with 0.63 mg/kg bw had signs of liver toxicity consistent with those seen in the highest dose group (approx 63 mg/kg bw). Based on results of the one-year study in dog (see below), this effect in one low-dose animal was not considered to be treatment-related.

In the 1-year dog study, the highest dose tested (2.8 mg/kg) was the no effect level. However this very low dose-level does not allow to make relevant conclusions about the NOAEL value.

In the 90-day rat study the NOEL was approximately 15 mg/kg bw/day, with liver toxicity being the primary toxic effect observed at the higher doses. This consistency between the two species in NOAELs indicates that they are equally sensitive, or that the Dog (a larger animal) is perhaps slightly less sensitive to the liver toxicity, since allometric differences should result in a lower NOEL in Dogs compared with Rats.

In rabbits, data relate to developmental and reproductive toxicity studies. One study performed with 4,4-dimethyloxazolidine (hydrolyzing in stomach into approx. 0, 2.2, 6.6, 22 mg/kg AMP) did not reveal any liver effects based on gross pathology or liver weights. In another study, 75 mg/kg AMP-95 induced mortality attributed to vacuolar degeneration/necrosis and multifocal hepatic necrosis; macrovesicular hepatocellular vacuolation was reported in all dose groups, whereas minimal microvesicular vacuolation was reported in the lowest dose group (9.5 mg/kg).

Given the available toxicokinetic information on AMP in the rat, and the consistency in toxic effects and NOAELs in rats, rabbits and dogs, it is proposed to use the NOAEL from the 2-generation study performed using the associated chemical 4,4-dimethyl oxazolidine. This substance hydrolyses almost immediately in the stomach following oral dosing; releasing formaldehyde and AMP (refer to summary of reproductive toxicity). In this study the rat the NOEL, the equivalent dose of AMP administered was 11 mg/kg bw/day. Again, the primary systemic toxic effect observed was hepatotoxicity at the next highest dose. The preference for using this study, and not the repeated dose rat or dog studies, is that the available toxicokinetic information allows a far more accurate extrapolation from the oral dose route used in the study to the dermal exposure route in humans when deriving a DNEL. With respect to extrapolating from the 2 -generation study to a long term human exposure situation, a factor of 1 is considered sufficient due to the length of this study, and the consistency of the results with the sub-chronic and chronic rat and dog studies.

Dermal route:

Two studies evaluated dermal exposure to AMP (Carney et al. 2005b; Thorsrud & Carney 2006). Both were developmental toxicity studies in which AMP was applied to the skin of rats from gestation days 6 through 20, up to 300 or 380 mg/kg/day equivalent pure AMP dose-levels. In both studies, no evidence of systemic maternal toxicity was noted, no statistically significant differences in mean liver weights or relative liver weights was reported, and no treatment related gross pathologic observations were reported. However, these studies did not include assessment of liver histopathology or clinical chemistry changes.

Local effects: 300 mg/kg/day of AMP produced skin scabbing and scaling. This was the basis for deriving a maternal toxicity NOAEL of 100 mg/kg/day.

Inhalation:

Exposure of rats to AMP for 6 hours/day for 5 consecutive days at target concentrations of 700, 1400, and 2000 mg/m3 (Low, Mid, and High, respectively) resulted in no early deaths.

In general, exposure to the test material resulted in reduced body weight at the Mid and High dose levels. Moderately increased monocyte count and aspartate aminotransferase and moderately decreased albumin were seen in the Mid and/or High dose groups. Statistically significant increases in mean relative organ weight were seen in adrenals (High dose males), liver (Mid and High dose males and females) and kidneys (Mid and High dose females). Minimal liver vacuolation was noted microscopically in Mid- and High-dose animals.

Local effects: Skin scabbing was noted at all dose-levels due to irritation caused by the test article. Local effects on skin and respiratory tract were confirmed post-mortem in Mid and High dose and to a lesser extent Low dose animals. This consisted in skin necrosis and ulceration, atrophy of goblet cells in nasal cavity, squamous metaplasia in respiratory and transitional epithelium and ulceration of the turbinates. A NOAEL could not be determined due to these local effects.

Overall conclusion across routes:

Oral route is the only route where significant liver toxicity is expressed, as the stomach's ability to cope with the alkalinity of the test item allows to reach high dose-levels, and because systemic absorption is also optimal. Liver toxicity was seen in rats, rabbits and dogs, but no severe fatty change was noted in any study.

By more realistic exposure routes i.e. inhalation and skin contact, significant adverse local effects (skin and upper respiratory tract) occur at concentrations where no or minimal liver effects are noted. As actual use patterns lead to effective control of local effects based on low in-use concentrations, closed systems and/or PPE, it is unlikely that any systemic effects would occur via these exposure routes.

Probable mode of action:

Choline and phosphatidyl choline are key in the transport of triglycerides from the hepatocytes in the form of VLDL (very low density lipoprotien). Interfering with the manufacture of phosphatidyl choline thus limits the capacity of the liver to transport lipids. The effect of AMP on the liver appears to be a result of interference with phospholipid synthesis in the hepatocytes. Various publications on AMP from the 1950's and 1960's have identified that it is capable of becoming incorporated into phospholipids in place of ethanolamine and/or choline and that it inhibits the uptake of choline by the liver cells. AMP also appears to inhibit the formation of choline in the liver via the conversion of ethanolamine to choline. It is possible that a phosphatidyl AMP moiety is competing for the enzyme phosphatidyl ethanolamine methyl transferase (converts phosphatidyl ethanolamine to phosphatidyl choline), preventing the formation of phosphatidyl choline, however it is as yet unclear as to the exact mechanism. What is however clear is that in the presence of suficient dietary choline, the effects of AMP on the liver are prevented, and this is likely due to the lower dependance on de novo choline synthesis when sufficient choline is present in the diet. Thus the hepatotoxicity is in part dependent on the presence of sufficient choline in the diet.

The transport of lipids from hepatocytes is fairly consistent throughout mammalian physiology, i.e. the use of a phospholipid based transport such as VLDL. Therefore the effects observed in rats, rabbits and dogs are likely relevant to man. However the increase in hepatic lipids is only the first step in the toxicity to the liver, and further indicators of toxicity are only evident after some accumulation of lipids. There is no evidence of liver toxicity (e.g. increased liver weight, increase in liver enzymes in the blood etc.) at doses where lipid accumulation in hepatocytes is not apparent . It is also apparent that the liver toxicity is the most sensitive endpoint, occurring at doses lower than those causing other effects, such as increased post implantation loss (refer to reproductive toxicity section).

AMP behaves as a competitive inhibitor or alternative agonist to one or more enzymes that bind and convert choline into PC that is important for VLDL production, leading to dysfunction of lipid transport, initial histological evidence of intracellular fat accumulation in vacuoles, and ultimately fatty liver accompanied by hepatocyte toxicity and pathologic changes. However, AMP-induced liver toxicity can only occur when high amounts of AMP are available to the liver relative to choline. This mode of action is consistent with animal studies using excess choline to reverse AMP effects as well as observations in humans of increased fatty liver effects among susceptible individuals who do not consume sufficient choline in their diet.

Justification for classification or non-classification

According to the GHS criteria for specific target organ toxicity (STOT RE), “morphological changes that are potentially reversible but provide clear evidence of marked organ dysfunction (e.g. severe fatty changes in the liver)” warrant classification.

The few systemic effects noted by dermal route and inhalation, the only routes relevant to worker risk asessment, clearly do not warrant any STOT RE classification.

Concerning systemic effects by oral route, none of the studies reported any severe fatty change. In addition, all available information on mode of action support a competition with choline, which can only occur when AMP is in excess of choline supply. Therefore, these toxic effects cannot occur at actual use levels because based on AMP levels in commercial products, AMP exposure is negligible compared to dietary supply of the essential nutrient choline. Absence of risks upon low-dose ingestion has been concluded by renowned regulatory bodies worldwide: US FDA approved AMP in various food contact applications (adhesives, coatings and fillers in paper/paperboard, certain resinous and polymeric coatings),  German BfR approved AMP in Recommendation XXXVI (paper and board for food contact),  AMP is in the positive list of Swiss Ordinance 817.023.21 (packaging inks) and was historically also in the positive list China GB 9685 (version 2008, additives for food contact materials).

Therefore, in the absence of severe hazard by any route, in the absence of risk with actual exposures, and in line with regulatory bodies for food contact assessments, it is concluded that STOT RE classification does not apply to AMP.