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1 -Diethylhydroxylamine toxicity was evaluated in a 28-day study in rats performed according to the OECD TG # 412 (Naas, 1996a). The test article was administered via nose-only inhalation to three groups, each comprised of 15 male and 15 female Crl: CDBR rats, for a period of six hours per day, five days per week, for four consecutive weeks (minimum of 20 total exposures). The targeted exposure concentrations were 15, 150 and 1500 ppm. The test atmosphere concentrations were monitored by infrared absorbance and were found to be 15, 150 and 1500 ppm (54.6, 546.0 and 5481.8 mg/m3). A concurrent control group of identical design received only filtered air, on a comparable regimen. The animals were observed for clinical signs and effects on body weight, food consumption and clinical pathology parameters. Data from detailed physical examinations, including Functional Observational Battery data (handling and open field observations), were recorded during the pretest period and during weeks 0 through 5. After completion of exposure, 5 rats/sex/group entered an approximate two-week (non exposure) recovery period, after which they were euthanized; necropsies were performed, and selected organs were weighed. The remaining rats in each group were euthanized immediately following the exposure period and necropsied as described above. A microscopic examination was conducted on selected tissues from all groups.In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies. The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level. The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related findings were noted during the Functional Observational Battery evaluations. Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animals were similar to the control group values. At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group mates and females, and the lymphocyte count was reduced in the females in this group. Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the mates in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm mates, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level. No test article-related internal fmdings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the 1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy. test article-related microscopic observations were noted. At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of non suppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal non suppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level.In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights-and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week non exposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues. Based on these results, exposure levels of 150 ppm (546.0 mg/m3) and 15 ppm (54.6 mg/m3) were considered to be the NOAEC (no observed adverse effect concentration) and NOEC (no observed effect concentration), respectively, for systemic toxicity and the exposure level of 15 ppm (54.6 mg/m3) was considered to be the NOEC for nasal irritation.In a range-finding study, diethylhydroxylamine was administered to three groups of 5 male and5 female CD rats by nose-only inhalation for a period of six hours per day, for five days (Naas, 1996b). Test atmosphere concentrations were monitored by infrared absorbance and were found to be 15, 149, 450 and 1541 ppm (target concentrations were 15, 150, 450 and 1500 ppm). A control group received filtered air. Urogenital matting was noted on the first day of exposure, only. Body weight gain and food consumption were slightly reduced at 1500 ppm throughout the week. Liver weights (absolute) were increased at 450 ppm and 1500 ppm in females (statistically significant at 1500 ppm), and mean weights were significantly increased at 450 ppm and 1500 ppm in females.Long-Evans hooded rats were exposed for at least 2 years by inhalationto9-27 ppm diethylhydroxylamine and the vapor of diethylamine hydrogen sulfite (Heicklen et al., 1981). In one of three test chambers each containing 45-49 rats, the rats were also exposed to 9 ± 2 ppm of nitroethane. In thefirst12 months of the experiment two males and two females from both the control chamber and the chamber containing all three gases were sacrificed at 3-month intervals. After the first year only moribund animals were sacrificed except at the very end of the study when all remaining animals were sacrificed. Although haematological and blood chemistry evaluations indicated no significant differences between the control and exposed animals, gross and microscopic pathology findings showed some variations, especially in the first year. Very early one test animal developed a hemangioendothelioma, but no additional ones developed later. Also hydrometra of the uterus, a condition common in old virgin female rats, was found in four exposed and one control female. Chronic tracheitis was found in five exposed and two control animals. Thyroid lesions were seen in the exposed animals after 6 months exposure, but not in animals exposed 9 months or longer. Examinations for animals exposed more than1 year indicated no significant differences between the control and test groups, except for interstitial cell tumors of the testes which showed up in 4 of the 47 exposed males that were examined comparedto0in the 25 control males. However, this incidence (8.5%) is too small to establish any definite conclusion.

2-Diethylhydroxylamine was evaluated in a 28-day repeated dose study on rats according to OECD 407 guideline (Shin Nippon, 2000). 5 males and 5 females were given the dose-levels at 20, 100 and 500 mg/kg/day . The control group was given purified water as a vehicle. A recovery group was was added for the high dose-level and the control. During the dosing and recovery periods , no animals died in any group. In clinical signs, salivation and reddih eye gum in males and females, reddih rhinorrhea in males and reddish urine in females werer observed transiently after dosing in the 500 mg/kg/day group. No abnormalities were observed in clinical signs in any animal during the recovery period. In general behaviour, salivation was observed in males in the 500 mg/kg/day group on day 27 of the dosing period. No abnormalities were observed during the recovery period. No test article related changes were noted in food consmuption, body weight, ophthalmology, urinalysis, hematology blood biochemistry, gross pathology, organ weight or histopathology.

It was concluded from these results that under the conditions of this study, the NOEL was 100 mg/kg/day and the NOAEL was 500 mg/kg/d in both males and females. Additionally, the changes that were noted during the dosing period were judged to be reversible.

3-The potential toxicity of the test item,N, N-DIETHYLHYDROXYLAMINE, was evaluated after daily oral administration (gavage) to rats at 50, 150 and 500 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period according to OECD 408 guideline (Modeste, 2018).

 Treatment-related clinical signs were limited to ptyalism at the high-dose only. Non-adverse treatment-related changes were seen at blood biochemistry and sperm analysis. There were no effects on body weight, food consumption, estrous cycles, hematology parameters, thyroid hormones and there were no test item-related ophthalmological findings.

 Non-adverse changes were observed at pathology in the kidneys, liver, adrenals, forestomach and stomach at 500 mg/kg/day, and in the kidneys and adrenals at 150 and 50 mg/kg/day.

 Under the experimental conditions of the study and based on the absence of adverse effects up to the high-dose, the No Observed Adverse Effect Level (NOAEL) in rats administered N, N-DIETHYLHYDROXYLAMINEfor 13 weeks was set at 500 mg/kg/day.

4 -Low level (9-27 ppm diethylhydroxylamine) inhalation exposure of rats for 2 years did not indicate any evidence of systemic toxicity and nasal irritation, however this study was complicated by the co-administration of nitroethane and/or diethylamine hydrogen sulfite

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

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:
09 March 2018 - 06 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
21 September 1998
Deviations:
yes
Remarks:
A number of deviations occured during without any impact on the integrity of the study.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age: on the first day of treatment, the animals were 5 to 6 weeks old
- Mean body weight: on the first day of treatment, the males had a mean body weight of 199 g (range: 169 g to 229 g) and the females had a mean body weight of 157 g (range: 137 g to 180 g)
- Fasting period before study: no
- Housing: the animals were housed in twos or threes from the same sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 11 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 26 March 2018 to 06 August 2018.
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Solution in the vehicle
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: Gas Chromatography with FID detection (GC-FID)
Test item concentrations:
Once in Weeks 1, 4, 8 and 12, all within the acceptance criterion.
A sample was taken from control and test item dose formulations and analyzed using the validated method
Acceptance criterion:
Measured concentration = nominal concentration ± 10%

Homogeneity: not assessed, formulation was a solution.
Checked parameters, acceptance criteria and obtained results are detailed in the validation report

Stability: 5 days at room temperature.
Duration of treatment / exposure:
13 weeks followed by a 6-week treatment-free period
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females per group (+ 5 males and 5 females for treatment-free period in control and high dose groups).
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for dose selection:
The dose levels were selected in agreement with the Sponsor, based on the results of previous toxicity studies: a 2-week dose-range finding study and a 28-day toxicity study (OECD 407), both performed in the same species.
In the 2-week dose-range finding study, three groups of Sprague-Dawley rats were treated with the test item at 100 mg/kg, 300 mg/kg and 1000 mg/kg by the oral route. At 1000 mg/kg/day, lower body weight gain was noted in males over the study period. Higher liver (+14% to +23%) and kidney (+10 to +22%) weights were recorded in both sexes when compared to controls. At 300 mg/kg/day, higher liver (+15%) and kidney (+10%) weights were noted in females only. At 100 mg/kg/day, no test item related changes were noted.

In the 28-day toxicity study, three groups of Sprague-Dawley rats received the test item at 20, 100 or 500 mg/kg/day by the oral route. No changes in body and organ weights were noted. At microscopic examination, the changes observed were considered to be incidental.

- Rationale for animal assignment: computerized randomization procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORTALITY/MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment and treatment-free periods, including weekends and public holidays.

CLINICAL SIGNS:
- Time schedule: Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs (this was performed in the afternoon on each day of FOB tests).

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

FUNCTIONAL OBSERVATION BATTERY (FOB):
- Time schedule: each main animal was evaluated once in Week 12 before the daily treatment (not for recovery animals).
This evaluation included a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli, and motor activity.

NEUROBEHAVIOURAL EXAMINATION:
Detailed clinical observation
The following parameters were assessed and graded:
- in the cage: "touch escape",
- in the hand: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis),
- in the standard arena (two-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.

Reactivity to manipulation or to different stimuli
The following parameters measurements, reflexes and responses were recorded:
- touch response,
- forelimb grip strength,
- pupillary reflex,
- visual stimulus response,
- auditory startle reflex,
- tail pinch response,
- righting reflex,
- landing foot splay,
- rectal temperature (at the end of the observation period).

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

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

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

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: ophthalmological examinations were performed on main animals (recovery animals excluded) before the beginning of the treatment period, on control and high-dose animals on one occasion at the end of the treatment period.
As a finding was observed at the end of the treatment period on one group 4 male, these examinations were carried out on low- and intermediate-dose animals (i.e. groups 2 and 3), at the discretion of the Study Director, and were also carried out on all recovery animals at the end of the treatment-free period.
The pupils of the animals were dilated with tropicamide (Mydriaticum®, Laboratoires Théa, Clermont Ferrand, France). After assessment of the corneal reflex (at instillation of the tropicamide), the appendages, optic media and fundus were examined by indirect ophthalmoscopy (Oméga 500, Heine, Herrsching, Germany).
A slit-lamp biomicroscope (Portable slit-lamp SL-17, Kowa, Japan) was used to investigate abnormalities of the anterior segment and the lens.

HEMATOLOGY:
- Time schedule: the parameters were determined for all main animals at the end of the treatment period.
- Bone marrow: two bone marrow smears were prepared from the femoral bone (at necropsy) of all animals euthanized on completion of the treatment or treatment-free period and stained with May Grünwald Giemsa.
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: all main animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY:
- Time schedule: the parameters were determined for all main animals at the end of the treatment period.
As some changes were noted on main animals at the end of the treatment period, these examinations were carried out on recovery animals (males and females) at the end of the treatment-free period.
- Animals fasted: Yes
- How many animals: all main animals
- Parameters checked in table [No. 2] were examined.

THYROIDS:
- Time schedule: An additional blood sample was taken from all animals euthanized at the end of the treatment period.
The levels of the thyroid hormones (T3 and T4) were determined by a LCMS-MS method and Thyroid Stimulating Hormone (TSH) was evaluated using a Luminex MAP technology. Analyses were performed on control-and high-dose main animals (groups 1 and 4) only.

URINALYSIS:
- Time schedule: the parameters were determined for all main animals at the end of the treatment period.
As some changes were noted on main animals at the end of the treatment period, these examinations were carried out on recovery animals (males and females) at the end of the treatment-free period.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table 5
The body weight of each animal was recorded before euthanasia at the end of the treatment or treatment free period. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

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


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

PREPARATION OF HISTOLOGICAL SLIDES
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable, embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except for the testes and epididymides which were stained with hematoxylin/PAS).
Kidneys from all males (main + recovery) were kept no longer than 96 hours in formalin (tissue intended for immunohistochemistry).

The tissue processing was performed at Citoxlab France.

One additional kidney slide of all main males euthanized at the end of the treatment period was immunostained with an antibody for Alpha-2-microglobulin protein.

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

In addition a detailed examination of the testes was performed for control-and high-dose males (groups 1 and 4) at the end of the treatment period, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin-PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
According to the microscopic results of the high-dose group, the following tissues from the low- (group 2) and intermediate-dose group (group 3) at the end of the treatment period and recovery groups at the end of the treatment free-period were examined:
- forestomach (males and females),
- kidneys (males and females),
- stomach (males),
- liver (males),
- adrenal glands (males).
Other examinations:
SPERM PARAMETERS
Before euthanasia at the end of the treatment period, each main male was injected intraperitoneally with sodium pentobarbital.
As some changes were noted on main animals at the end of the treatment period, these examinations were carried out on recovery animals at the end of the treatment-free period (epididymal sperm motility, morphology count, testicular sperm count).

EPIDIDYMAL SPERM
Under deep anesthesia and after epididymis weight, sperm from the cauda of the left epididymis was sampled for motility and morphology investigations. Animals were then euthanized.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.

EPIDIDYMAL SPERM MOTILITY (all main males)
The sperm was evaluated on a slide. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.

EPIDIDYMAL SPERM MORPHOLOGY (all main males)
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Smears were prepared for all main males. Morphology was evaluated on groups 1 and 4. As some changes were noted on group 1 and 4 animals at the end of the treatment period, morphology was also evaluated on intermediate-dose groups and on recovery animals.

Results were expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

EPIDIDYMAL SPERM COUNT (all main males)
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

TESTICULAR SPERM (all main males)
The left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (using a time divisor of 6.10).

MONITORING OF ESTROUS CYCLE
The estrous cycle stage was determined daily on all main females from a fresh vaginal lavage (stained with methylene blue), daily for 21 consecutive days at the end of the treatment period.

According to the results obtained on principal females at the end of the treatment period, these examinations were carried out daily on recovery females for 21 consecutive days before the end of the treatment-free period.
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, seminology, hematology, blood biochemistry, urinalysis and thyroid hormone data according to the following sequence.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Ptyalism was observed over the treatment period in 15/15 males and 15/15 females at 500 mg/kg/day and occasionally in 1/10 males and 1/10 females at 150 mg/kg/day. This clinical sign, non-adverse and commonly observed when a test item is given by gavage, was attributed to the taste/texture of the test item formulation at high concentration and was no longer observed during the treatment-free period.

The other clinical signs observed during the study, such as thinning of hair, scabs, alopecia, hunched posture, soft feces, soiled parts of the body, reflux at dosing, exophthalmos were not considered to be test item-related since they were reported sporadically in only a few animals, observed in both control and test item-treated animals, observed without a dose-relationship and/or can commonly be observed in laboratory rats of this strain and age.
Mortality:
no mortality observed
Description (incidence):
No unscheduled deaths occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on mean body weight and body weight change during the study.

Occasional statistical differences from controls were transiently observed in mean body weight gain at all dose levels but they were considered to be consistent with normal biological variation and therefore incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects on mean food consumption throughout the study.

The few differences in mean food consumption recorded between control and test item-treated animals, including those statistically significant, were not attributed to the test item treatment as they were of minimal magnitude remaining within the range of variation commonly observed in rats, not dose-related or consistent with spillage.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related ophthalmological changes at the end of the treatment or treatment-free period.
Vitreous hemorrhage was observed at the end of the treatment period in the right eye of 1/10 males given 500 mg/kg/day. This finding was observed unilaterally in only one animal and no related finding was seen at microscopic examination. Given the low incidence and absence of lesion at histology, this was considered to be incidental.

Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects among hematology parameters at the end of the treatment period.

Any apparent differences between control and test item-treated animals were considered to be incidental and not test item-related since they were isolated, consistent with normal biologic variation, observed with no dose-relationship and/or negligible in magnitude.

Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See Table 1
When compared with controls, a slight decrease in chloride concentration and a slight increase in potassium concentration were noted in animals administered at 500 mg/kg/day.
Slightly higher mean cholesterol and triglycerides levels were also observed in both genders at the same dose level.
These changes were considered to be test item-related but not adverse given their slight magnitude. They were no longer observed at the end of the treatment-free period (opposite trend for triglyceride concentration in males).

Other apparent differences between control and test item-treated animals were considered to be incidental and not test item-related since they were isolated, consistent with normal biological variation, observed with no dose-relationship and/or negligible in magnitude.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in urine volume was noted in males at = 150 mg/kg and in females at 500 mg/kg. In the high-dose group, this correlated with tubular vacuolation and dilatation seen at microscopic examination.

Statistically significant higher mean urine volume was observed at the end of the treatment period in males given 150 mg/kg/day and in males and females given 500 mg/kg/day (up to 19 mL in males vs. 9 mL in controls; 11 mL in females vs. 7 mL in controls). In the high-dose group, this correlated with tubular vacuolation and dilatation seen at microscopic examination.

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no test item-related effects at the detailed clinical examination, assessment of reactivity to manipulation and different stimuli and motor activity evaluation at the end of the treatment period.
No ptyalism was observed on test item-treated animals at detailed clinical examination as part of the FOB although it was regularly recorded at least for the high-dose group. This may be explained by the fact that clinical signs are recorded after test item administration, whereas the FOB was performed before the daily treatment (suggesting transient ptyalism after each daily treatment).

Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See Table 2
At the end of the treatment period
Increased absolute and relative-to-body kidney and liver weights were noted in females treated at 500 mg/kg/day (p<0.05 or 0.01). A similar trend was noted for the relative-to-body weights of liver in males treated at 500 mg/kg/day.

The increased liver weights correlated with the hepatocellular hypertrophy seen in 2/10 males treated at 500 mg/kg/day at microscopic examination. Although there were no microscopic correlates in females, this difference was considered to be related to the test item administration.
The increased kidney weights correlated with microscopic tubular vacuolation and dilatation seen mainly in females.
Increased absolute and relative-to-body adrenal gland weights were noted in males treated at 500 mg/kg/day (p<0.05). These differences correlated with microscopic cortical vacuolation.
The other differences were considered to be unrelated to the test item administration since they were not dose-related, of minimal magnitude and/or had no microscopic correlates.

See Table 3
At the end of the treatment-free period
Increased absolute and relative-to-body adrenal gland weights were noted in males previously treated at 500 mg/kg/day (p<0.05). Although these differences had no microscopic correlates, they were considered to be related to test item administration.
Minimally increased absolute and relative-to-body liver weights were noted in females previously treated at 500 mg/kg/day (p<0.05). In the absence of test item-related changes at the end of the treatment period and in view of their low magnitude, these differences were considered to be fortuitous.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period
Thickened mucosa was noted in the forestomach from 1/10 females treated at 500 mg/kg/day and white discoloration of forestomach was recorded in another female. These changes correlated with microscopic hyperkeratosis (thickening and white discoloration) and fibroplasias and hyperplasia of squamous cells (white discoloration). Although seen with incidence, these changes were considered to be related to the test item administration.

The other changes were considered to be part of the spontaneous background in the rats of these strain and age.

At the end of the treatment-free period
There were no test item-related findings.
The changes were considered to be part of the spontaneous background in the rats of these strain and age.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See Table 4

At the end of the treatment period
. Kidney
Test item-related minimal to slight cortical tubular vacuolation, dilatation and hyaline casts (homogeneous eosinophilic deposits in the lumen of medullary tubules) were noted in males and females treated at 500 mg/kg/day and, to a lesser extent at 50 and 150 mg/kg/day in females. In addition, minimal infiltrate of mononuclear cells in the interstitium was noted.
In the absence of degeneration/necrosis and in view of the low magnitude of these changes, they were considered to be non-adverse.
It is noteworthy that the immunohistochemistry staining for a2-µglobulin did not reveal any differences between test item-treated animals and controls.

. Liver
Minimal centrilobular hepatocellular hypertrophy was noted in 2/10 males treated at 500 mg/kg/day.
In view of the very low incidence and magnitude, this test item-related finding was considered to be non adverse.

. Forestomach and stomach
Test item-related minimal to slight, focal or multifocal vacuolar degeneration, hyperkeratosis, squamous cell hyperplasia, submucosal edema, fibroplasia, mixed or mononuclear cell infiltrate and/or hemorrhage were noted in males and females treated at 500 mg/kg/day and, to a lesser extent, hyperplasia and hyperkeratosis at 50 or 150 mg/kg/day.
In the absence of ulceration affecting the mucosa and given their low magnitude, these findings were considered to be non-adverse.

There was a focal minimal degeneration/necrosis in the stomach from one high-dose male, with equivocal significance in view of the isolated occurrence of this change.

. Adrenal glands
Test item-related minimal to slight cortical vacuolation was noted in 1/10 males treated at 50 mg/kg/day, 3/10 males treated at 150 mg/kg/day and in 7/10 males treated at 500 mg/kg/day.
In the absence of degeneration/necrosis and in view of its low magnitude, this change was considered to be non-adverse.
This correlated with the increased weights at the high-dose level.


. Vagina
Slight to moderate mucification was noted in the vagina from 2/10 females treated at 500 mg/kg/day.
This non-adverse change may be secondary to stress and was related to the test item administration. In view of the low incidence of this change, it was considered to be of minor toxicological importance.

No noteworthy findings were noted in testes and epididymides.

The other isolated findings were considered not to be related to the test item administration as they were not dose-related, were of low magnitude and/or commonly seen in the untreated rat kept under laboratory condition.

At the end of the treatment-free period
In the forestomach or kidneys (males and females), stomach, liver or adrenal glands (males) examined at the end of the treatment-free period, there were no test item-related changes, suggesting complete reversibility of the test item-related microscopic changes after the recovery period.
Histopathological findings: neoplastic:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Estrous cycles - At the end of the treatment period
See Table 5
In all groups including control females, several animals presented irregular cycles, delayed transiently at the diestrus stage or until the end of the evaluation. This led to a high inter-individual variability in group 1 and even more in group 3 animals.
Overall, the number of cycles was slightly higher in the high-dose group (500 mg/kg/day), linked to slightly lower cycle length when compared to control females. However, females in group 4 seemed to be less affected by the irregularity in cycle length and it was considered that the changes were not related to the test item administration.
The statistically significant higher mean cycle length noted at 150 mg/kg/day was due to 3/10 females (blockage in diestrus for at least 13 days). Blockage in diestrus was also observed in the other groups including controls even if this was for a lesser duration. In absence of dose-relationship, this finding was considered to be incidental.

At the end of the treatment-free period
See Table 6
In group 4 females, the cycle length was slightly higher than in control animals (contrary to the observation made at the end of the treatment period), due to 3/5 females either delayed in diestrus or with several consecutive days in diestrus before starting a new cycle. Taking into account that control females were also affected with 2/5 females delayed in the diestrus stage, a relationship with the test item administration was considered unlikely.

Seminology
At the end of the treatment period
See Table 7

There were no test item-related effects on mean epididymal sperm motility.

At 150 and 500 mg/kg/day, there were dose-related slightly lower mean percentages of morphologically normal epididymal sperm [due to dose-relatedly higher mean percentages of abnormal sperm flagellum (abnormal + no flagellum)], lower mean number of epididymal and testicular sperm as well as lower daily sperm production rate. The trend towards higher mean percentage of sperm with abnormal flagellum was also seen at 50 mg/kg/day.
In test item-treated groups, some individual data were lower/higher than the 5%/95% percentile of the Historical Control Data generally in a dose-related manner, while this was not observed in controls.

Given the dose-relationship of these findings, a test item effect was considered to be likely, however, the magnitude of the changes being low and in absence of test item-related histopathological findings in the right testis and epididymis at the high-dose, the changes were considered non-adverse.

At the end of the treatment-free period
At the end of the 6-week treatment-free period, all sperm analysis data in males previously given 500 mg/kg/day were similar to controls, generally within Historical Control Data and considered as reversible, except for epididymal sperm count which was still slightly but not statistically lower than controls (375.9 vs. 459.8 106/g cauda: -18%) and within Historical Control Data.

Thyroid hormones
There were no test item-related changes on mean T3, T4 or TSH levels measured on control and high dose animals at the end of the treatment period.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

Table 1

Blood biochemistry 

Sex

Male

Female

Dose level (mg/kg/day)

0

50

150

500

0

50

150

500

End of treatment period

 

 

 

 

 

 

 

 

Potassium (mmol/L)

3.88

3.87

4.10

4.20**

3.60

3.61

3.84

4.02**

 

-

0

+6

+8

-

0

+7

+12

Chloride (mmol/L)

105.8

106.9*

106.3

103.9**

105.3

106.1

106.0

102.8**

 

-

+1

0

-2

-

+1

+1

-2

Cholesterol (mmol/L)

1.54

1.52

1.46

1.86*

2.02

2.12

2.02

2.70**

 

-

-1

-5

+21

-

+5

0

+34

Triglycerides (mmol/L)

0.45

0.40

0.37

0.70*

0.36

0.29

0.33

0.63**

 

-

-11

-18

+56

-

-19

-8

+75

End of treatment-free period

Potassium (mmol/L)

3.87

-

-

3.93

3.74

-

-

3.84

 

-

 

 

+2

-

 

 

+3

Chloride (mmol/L)

105.6

-

-

106.9

106.5

-

-

106.9

 

-

 

 

+1

-

 

 

0

Cholesterol (mmol/L)

1.71

-

-

1.63

2.06

-

-

2.47

 

-

 

 

-5

-

 

 

+20

Triglycerides (mmol/L)

0.64

-

-

0.38**

0.38

-

-

0.47

 

-

 

 

-41

-

 

 

+24

-: not applicable.

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

Table 2

Organ weights

At the end of the treatment period

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose level (mg/kg/day)

50

150

500

50

150

500

Number of examined animals

10

10

10

10

10

10

- Final body weight

0

+5

-2

-1

0

-2

- Adrenal glands

. absolute

-4

-1

+15*

-9

-6

-3

. relative-to-body

-3

-6

+16*

-8

-7

-1

- Kidneys

. absolute

+1

+4

+4

+3

+2

+12*

. relative-to-body

+1

-1

+6

+4

+1

+14**

- Liver

. absolute

+3

+3

+12

+5

+4

+18*

. relative-to-body

+3

-1

+14**

+5

+3

+20**

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

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

Table 3

At the end of the treatment-free period

 

Sex

Male

Female

Group

4

4

Dose level (mg/kg/day)

500

500

Number of examined animals

5

5

- Final body weight

-1

+4

- Adrenal glands

. absolute

+18*

-2

. relative-to-body

+19*

-6

- Liver

. absolute

-2

+13*

. relative-to-body

-1

+8*

Statistically significant from controls: *: p<0.05.

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

Table 4

Microscopic examination

 

Sex

Male

Female

Group

1

2

3

4

1

2

3

4

Dose level (mg/kg/day)

0

50

150

500

0

50

150

500

Number of animals per group

10

10

10

10

10

10

10

10

Kidney; tubular vacuolation

. grade 1

-

-

-

-

-

2

1

7

Kidney;tubular dilatation

. grade 1

-

-

-

1

-

2

3

6

. grade 2

-

-

-

-

-

-

-

4

Kidney; hyaline casts

. grade 1

-

-

-

5

-

2

1

6

. grade 2

-

-

-

-

-

-

-

1

Liver; hepatocellular hypertrophy

. grade 1

-

-

-

2

-

na

na

-

Forestomach;vacuolar degeneration

. grade 1

-

-

-

-

-

-

-

1

. grade 2

-

-

-

1

-

-

-

-

Forestomach;hyperkeratosis

. grade 1

-

2

-

4

-

1

2

1

. grade 2

-

-

-

1

-

-

-

2

Forestomach;squamous cell hyperplasia

. grade 1

-

-

1

2

-

1

2

-

. grade 2

-

-

-

1

-

-

-

2

Forestomach;submucosal edema

. grade 2

-

-

-

1

-

-

-

1

Forestomach;fibroplasia

. grade 2

-

-

-

-

-

-

-

1

Adrenal;cortical vacuolation

. grade 1

-

1

3

5

-

na

na

-

. grade 2

-

-

-

2

-

na

na

-

Vagina;mucification

. grade 2

na

na

na

na

-

na

na

1

. grade 3

na

na

na

na

-

na

na

1

na: not applicable; -: not observed.

 

Table 5

Estrous cycle

End of treatment period

Dose level (mg/kg/day)

0

50

150

500

 

Treatment period

Number of cycles

3.3

3.6

2.8

4.2

SD

0.8

1.2

1.5

1.0

Cycle length (days)

4.8

4.0

8.1*

3.7

SD

1.4

0.3

5.5

0.4

Rats cycling normally

7

9

4

5

Cycle length = proestrus or estrus starts the estrous cycle until the day prior to the next non-consecutive estrus or proestrus. *: p<0.05.

A normally cycling is defined as having a mean average cycle of between 4 and 5 days.

 

Table 6

 

End of treatment-free period

 

Dose level (mg/kg/day)

0

500

 

Treatment-free period

Number of cycles

3.0

2.6

SD

1.9

1.5

Cycle length (days)

4.2

5.9

SD

0.4

2.6

Rats cycling normally

5

3

 

Table 7

Seminology

Dose level (mg/kg/day)

0

50

150

500

Historical data

% of motile epididymal sperm

97.3

99.3

99.4

96.8

[85.8 - 100.0]

 

-

+2%

+2%

-1%

% of morphologically normal epididymal sperm

97.6

96.1

93.6**

92.4**

[92.0 - 100.0]

 

-

-2%

-4%

-5%

% of sperm with abnormal flagellum

0.2

2.2*

4.5**

0.1

[0.00 - 2.00]

 

-

x11 fold

x22.5 fold

-50%

 

% of sperm with no flagellum

2.2

1.6

1.9

7.5*

[0.00 - 5.00]

 

-

-27%

-14%

x3.4 fold

 

Mean number of epididymal sperm (106/g cauda)a

458

463

375*

359*

[302.9 - 601.2]

 

-

+1%

-18%

-22%

Mean number of testicular sperm heads (106/g testis)a

130

123

106*

101**

[100.7 - 157.8]

 

-

-5%

-18%

-22%

Daily sperm production rate (106/g testis/day)

21.3

20.2

17.4*

16.6*

[16.5 - 25.9]

 

-

-5%

-18%

-22%

a: values rounded to three significant digits; -: not applicable.

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

Historical Control Data show the individual data percentiles [5%-95%].

 

Conclusions:
The potential toxicity of the test item was evaluated after daily oral administration (gavage) to rats at 50, 150 and 500 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period.

Treatment-related clinical signs were limited to ptyalism at the high-dose only.
Non-adverse treatment-related changes were seen at blood biochemistry and sperm analysis.
There were no effects on body weight, food consumption, estrous cycles, hematology parameters, thyroid hormones and there were no test item-related ophthalmological findings.

Non-adverse changes were observed at pathology in the kidneys, liver, adrenals, forestomach and stomach at 500 mg/kg/day, and in the kidneys and adrenals at 150 and 50 mg/kg/day.

Under the experimental conditions of the study and based on the absence of adverse effects up to the high-dose, the No Observed Adverse Effect Level (NOAEL) in rats administered for 13 weeks was set at 500 mg/kg/day.
Executive summary:

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

 

Methods

 

Three groups of ten male and ten female Sprague-Dawley rats were treated daily by the oral route (gavage) with the test item at 50, 150 or 500 mg/kg/day for 13 weeks. The test item was administered as a solution in the vehicle (water for injection) using a constant dose volume of 5 mL/kg/day. A control group of ten animals per sex received the vehicle alone, under the same experimental conditions. Recovery animals (five per sex) were included in the control-and high-dose groups and were kept for a 6-week treatment-free period on completion of the treatment period, to evaluate the reversibility of any findings.

 

Actual test item concentrations in the dose formulations administered in Weeks 1, 4, 8 and 12 were determined using a validated GC-FID analytical method.

The animals were checked daily for mortality and clinical signs. Detailed clinical examinations were performed weekly. A Functional Observation Battery (FOB) including a detailed clinical examination, the assessment of reactivity to manipulation and different stimuli as well as motor activity evaluation was performed on main animals in Week 12 before the daily treatment.

Body weight was recorded once before the beginning of the treatment period, on the first day of treatment, and then at least once weekly until the end of the study. Food consumption was recorded weekly.

Ophthalmological examinations were performed on all main animals before the beginning and at the end of the treatment period and on recovery animals at the end of the treatment-free period.

The estrous cycle was monitored (fresh vaginal lavage stained with methylene blue) for 21 consecutive days in all main females at the end of the treatment period and in all recovery females at the end of the treatment-free period.

Hematology, blood biochemistry and urinary investigations were performed on all main animals at the end of the treatment period. Blood biochemistry and urinary investigations were also performed at the end of the treatment-free period.

Determination of thyroid hormones (T3 and T4) and Thyroid Stimulating Hormone (TSH) levels was performed on blood samples collected on control-and high-dose animals at the end of the treatment period.

Seminology (epididymal sperm motility, morphology, count and testicular sperm count) was performed on all main and recovery males.

On completion of the treatment and treatment-free period, the animals were euthanized and a full macroscopic post-mortem examination was performed. Designated organs were weighed and selected tissue specimens were preserved. A microscopic examination was performed on a full list of tissues from control-and high-dose main animals euthanized at the end of the treatment period, and on target tissues from low- and intermediate-dose animals and recovery animals. In addition, specific stained slides of testes (hematoxylin-PAS) from control-and high-dose main males and of kidney (immunostained with an antibody for Alpha-2-microglobulin protein) from all main males were processed and examined.

 

Results

Actual test item concentrations in the analyzed dose formulations were -1.9 to +10.0% of nominal concentrations, thus within the accepted range (theoretical concentration ± 10%). No test item was detected in the control dose formulations.

 

No unscheduled deaths occurred during the study.

 

Ptyalism was observed over the treatment period in animals given 500 mg/kg/day and was no longer observed during the treatment-free period. It was attributed to the taste/texture of the test item formulation at a high concentration. There were no other test item-related clinical signs or changes among parameters evaluated at the FOB.

 

Body weight and food consumption were unaffected by the test item-treatment.

 

No test item-related ophthalmological findings were observed.

 

Despite high irregularity observed in the duration of cycles in all groups including controls, there were no changes in the estrous cycles considered to be linked to the test item administration.

 

Hematology parameters and thyroid hormones were unaffected.

Minor changes were seen at blood biochemistry (slightly lower chloride, and slightly higher potassium, cholesterol and triglycerides levels) in males and females at = 150 mg/kg/day. These changes were of low magnitude, reversible and were therefore not considered to be adverse.

 

Slightly higher urine volume was noted in males from 150 mg/kg/day and in females at 500 mg/kg/day, and correlated with non-adverse tubular vacuolation and dilatation seen at microscopic examination.

 

At sperm analysis, motility was unaffected but there were dose-related slightly lower percentage of morphologically normal epididymal sperm, lower number of epididymal and testicular sperm as well as lower daily sperm production rate at 150 and 500 mg/kg/day; dose-related higher mean percentages of sperm with abnormal or no flagellum was noted at all three dose levels. The magnitude of the changes being low and in absence of test item-related histopathological findings in the right testis and epididymis at the high-dose, none of these findings were considered to be adverse. They were fully reversible at the end of the 6-week treatment-free period, except for epididymal sperm count which still remained slightly lower than in controls.

 

Test item administration at 500 mg/kg/day induced non-adverse microscopic changes in the kidneys (microscopic tubular vacuolation and dilatation, hyaline casts), liver (centrilobular hepatocellular hypertrophy) and adrenals (cortical vacuolation) which correlated with increases in organ weights. Changes were also recorded in the forestomach (thickened mucosa and white discoloration correlating with non-adverse hyperkeratosis, fibroplasia and/or hyperplasia of squamous cells in females, vacuolar degeneration, submucosal edema, mixed or mononuclear cell infiltrate and/or hemorrhage in both genders) and in the stomach of one male (doubtful focal minimal degeneration/necrosis). These changes in the forestomach and in the stomach might be linked to the pH of the dose formulations (between 10 and 11).

 

None of these changes were seen at the end of the treatment-free period, with the exception of increased adrenal gland weights that was still noted in males but with no microscopic correlates.

 

At 150 and 50 mg/kg/day, non-adverse microscopic cortical tubular vacuolation, dilatation and hyaline casts were noted in the kidneys of females, together with adrenal vacuolation in all (at 150 mg/kg/day) or one male (at 50 mg/kg/day) and hyperkeratosis and/or hyperplasia of squamous cells in males and/or females.

Conclusion

The potential toxicity of the test item was evaluated after daily oral administration (gavage) to rats at 50, 150 and 500 mg/kg/day for 13 weeks, followed by a 6-week treatment-free period.

Treatment-related clinical signs were limited to ptyalism at the high-dose only.

Non-adverse treatment-related changes were seen at blood biochemistry and sperm analysis.

There were no effects on body weight, food consumption, estrous cycles, hematology parameters, thyroid hormones and there were no test item-related ophthalmological findings.

Non-adverse changes were observed at pathology in the kidneys, liver, adrenals, forestomach and stomach at 500 mg/kg/day, and in the kidneys and adrenals at 150 and 50 mg/kg/day.

Under the experimental conditions of the study and based on the absence of adverse effects up to the high-dose, the No Observed Adverse Effect Level (NOAEL) in rats administered for 13 weeks was set at 500 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Experimental exposure time per week (hours/week):
7
Species:
rat

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Age: seven and one-half weeks old at initiation of exposure
- Weight at study initiation: 197 to 241 grains for the males and from 148 to 189 grams for the females
- Fasting period before study: not relevant
- Housing: individually in clean suspended wire-mesh cages suspended above cage-board
- Diet (ad libitum): Purina® Certified Rodent Chow® #5002
- Water (ad libitum): Tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70° to 77°F
- Humidity (%): 22-56
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: In-Tox Products, Inc. nose-only exposure units
- Method of holding animals in test chamber: individually in tubes
- System of generating vapors: vaporization of the neat test material, using a glass U-tube filled with 3 mm glass beads
- Temperature, humidity, pressure in air chamber: Engineering probes and transmitters were used to monitor temperature (°C) and relative humidity (% RH). Oxygen content was measured by Mine Safety Appliances Remote Sampling Systems with an oxygen sensor. Negative pressure was monitored by Dwyer Magnehelic® Indicating Transmitter pressure gauges (0-2.0 inches of water).
- Air change rate: at least 12 to 15 air changes per hour
- Method of particle size determination:
- Treatment of exhaust air: drawing the air through an acetone dry ice bath and an in-lire HEPA filter

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectroscopy using a Miran lA gas analyzer
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test atmosphere sampling for concentration analysis was performed at approximate 60 minute (minimum) intervals during the exposure periods.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours per day , 5 days per week
Dose / conc.:
15 ppm
Dose / conc.:
150 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Ye
- Time schedule: twice daily for all animais, once prior to placement in the tubes and once approximately one hour after completion of exposure. The clinical condition of the animals also was monitored (to the extent possible for rats restrained in exposure tubes) during exposure periods. On nonexposure days, including the recovery period, clinical examinations were performed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning one week prior to initiation of test article exposure, and just prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet)
Differential Leukocyte Count*:
- Unsegmented Neutrophil (Unsegmented)
- Lymphocyte
- Monocyte
- Eosinophil -Basophil
- Segmented Neutrophil
(Segmented) RBC Morphology, Platelet Estimate
* Performed on the control and high dose groups at the week 4 and 6 necropsies and on the mid dose group at the week 4 necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Glucose, Urea Nitrogen, Creatinine, Total Protein, Albumin, Albumin/Globulin Ratio (A/G Ratio), Globulin, Calcium, Phosphorus, Chloride, Total Bilirubin, Gamma Glutamyltransferase, Bile Acids, Serum Aspartate Aminotransferase, Serum Alanine Aminotransferase, Serum Alkaline Phosphatase, Sodium, Potassium, Total Cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 or week 6 necropsies
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked:
Volume, Color (CLOR), Appearance (APP), Specific Gravity (SG), pH, Leukocytes (WBC), Nitrites (NIT), Protein (PRO), Glucose (GLU), Ketones (KET,) Bilirubin (BIL), Occult Blood (BLD), Urobilinogen, Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly, beginning one week prior to initiation of test article exposure, and prior to the scheduled necropsy
- Dose groups that were examined: all
- Battery of functions tested: handling observations, open-field observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.

ORGAN WEIGHTS: Yes
Adrenals Spleen
Brain Ovaries
Epididymides Testes
Heart Thymus Kidneys
Liver
Lungs

HISTOPATHOLOGY: Yes
Adrenals (2), Aorta, Bone with marrow (sternebrae), Brain (forebrain, midbrain, hindbrain), Eyes with optic nerve (2), Gastrointestinal tract ( Esophagus, Stomach, Duodenum, Jejunum, Ileum, Peyers' patches, Cecum, Colon, Rectum), Heart, Kidneys (2), Liver (sections of two lobes), Lungs [including bronchi, fixed by inflation with fixative (2)], Lymph node (bronchial, mesenteric, suprapharyngeal), Mammary gland (females only), Nasal cavities, Ovaries with oviducts (2), Pancreas, Parathyroids (if present), Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands [submaxillary (2)], Seminal vesicles (2), Skeletal muscle (vastus medialis), Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes with epididymides (2), Thymus, Thyroids, Trachea, Urinary bladder, Uterus with vagina, All gross lesions.
Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the treatment groups to the vehicle control group by sex. All means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a Digital® MicroVAX® 3400 computer with appropriate programming. Body weight, body weight change, food consumption, clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance followed by Dunnett's test.
Clinical laboratory values for cell types that occur at a low incidence (monocytes, eosinophils, basophils, unsegmented neutrophils) were not subjected to statistical analyses.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (Tables 1 through 6)
In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies.
The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level.

BODY WEIGHT AND WEIGHT GAIN (Tables 35, 36)
Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. 

FOOD CONSUMPTION (Table 37)
Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animals were similar to the control group values.

HAEMATOLOGY (Tables 38, 39, 40)
At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group mates and females, and the lymphocyte count was reduced in the females in this group.

CLINICAL CHEMISTRY (Table 41)
Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the mates in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm mates, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level.

URINALYSIS (Tables 42, 43)
Total urine volume was increased in the 1500 ppm group males and females relative to the control group values at the week 4 and week 6 evaluations; however, the increases were not statistically significant. In addition, urine volume is a highly variable parameter, and no other findings were noted in these animals that would account for this increase in volume; therefore, the changes in urine volume were not attributed to test article exposure.

NEUROBEHAVIOUR (Tables 7 through 20)
The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related findings were noted during the Functional Observational Battery evaluations

ORGAN WEIGHTS (Tables 47, 48, 49, 50)
At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the 1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy.

GROSS PATHOLOGY (Tables 44, 45, 46)
No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. 

HISTOPATHOLOGY: NON-NEOPLASTIC (Tables 51, 52, 53)
At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal nonsuppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level.
Dose descriptor:
LOAEC
Remarks:
systemic toxicity
Effect level:
1 506 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight reversible decreases in albumin and A/G ratio (females only)
Dose descriptor:
NOEC
Remarks:
systemic toxicity
Effect level:
15 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
LOAEC
Remarks:
nasal irritation
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis
Dose descriptor:
NOEC
Remarks:
nasal irritation
Effect level:
15 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis
Critical effects observed:
yes
Lowest effective dose / conc.:
150 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
other: liver and thymus
Organ:
liver
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights-and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues.
Executive summary:

Potential subchronic toxic effects of the test article, Diethylhydroxylamine (DEHA), were evaluated in this 28-day study in rats. The test article was administered via nose-only inhalation to three groups, each comprised of 15 male and 15 female Crl:CD®BRrats, for a period of six hours per day, five days per week, for four consecutive weeks (minimum of 20 total exposures). The targeted exposure concentrations were 15, 150 and 1500 ppm. The test atmosphere concentrations were monitored by infrared absorbance and were found to be 15, 150 and 1506 ppm. A concurrent control group of identical design received only filtered air, on a comparable regimen. The animals were observed for clinical signs and effects on body weight, food consumption and clinical pathology parameters. Data from detailed physical examinations, including Functional Observational Battery data (handling and open field observations), were recorded during the pretest period and during weeks 0 through 5. After completion of exposure, 5 rats/sex/group entered an approximate two-week (nonexposure) recovery period, after which they were euthanized; necropsies were performed, and selected organs were weighed. The remaining rats in each group were euthanized immediately following the exposure period and necropsied as described above. A microscopic examination was conducted on selected tissues from all groups.

In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies. The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level. The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related fmdings were noted during the Functional Observational Battery evaluations.Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animais were similar to the control group values. At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group males and females, and the lymphocyte count was reduced in the females in this group. Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the males in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm males, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level. No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy. At the necropsy of animals that died during the study, no test article-related microscopic observations were noted. At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal non suppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level. In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues. No toxicity was noted at a dose level of 15 ppm. Based on these results, exposure levels of 150 and 15 ppm were considered to be the NOAEC (no observed adverse effect concentration) and NOEC (no observed effect concentration), respectively, for systemic toxicity and the exposure level of 15 ppm was considered to be the NOEC for nasal irritation, under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
546 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Age: seven and one-half weeks old at initiation of exposure
- Weight at study initiation: 197 to 241 grains for the males and from 148 to 189 grams for the females
- Fasting period before study: not relevant
- Housing: individually in clean suspended wire-mesh cages suspended above cage-board
- Diet (ad libitum): Purina® Certified Rodent Chow® #5002
- Water (ad libitum): Tap water
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70° to 77°F
- Humidity (%): 22-56
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
inhalation: vapour
Type of inhalation exposure:
nose only
Vehicle:
clean air
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: In-Tox Products, Inc. nose-only exposure units
- Method of holding animals in test chamber: individually in tubes
- System of generating vapors: vaporization of the neat test material, using a glass U-tube filled with 3 mm glass beads
- Temperature, humidity, pressure in air chamber: Engineering probes and transmitters were used to monitor temperature (°C) and relative humidity (% RH). Oxygen content was measured by Mine Safety Appliances Remote Sampling Systems with an oxygen sensor. Negative pressure was monitored by Dwyer Magnehelic® Indicating Transmitter pressure gauges (0-2.0 inches of water).
- Air change rate: at least 12 to 15 air changes per hour
- Method of particle size determination:
- Treatment of exhaust air: drawing the air through an acetone dry ice bath and an in-lire HEPA filter

TEST ATMOSPHERE
- Brief description of analytical method used: infrared spectroscopy using a Miran lA gas analyzer
- Samples taken from breathing zone: yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test atmosphere sampling for concentration analysis was performed at approximate 60 minute (minimum) intervals during the exposure periods.
Duration of treatment / exposure:
28 days
Frequency of treatment:
6 hours per day , 5 days per week
Dose / conc.:
15 ppm
Dose / conc.:
150 ppm
Dose / conc.:
1 500 ppm
No. of animals per sex per dose:
15
Control animals:
yes, sham-exposed
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Ye
- Time schedule: twice daily for all animais, once prior to placement in the tubes and once approximately one hour after completion of exposure. The clinical condition of the animals also was monitored (to the extent possible for rats restrained in exposure tubes) during exposure periods. On nonexposure days, including the recovery period, clinical examinations were performed once daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning one week prior to initiation of test article exposure, and just prior to the scheduled necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Total Leukocyte Count (White Cell), Erythrocyte Count (Red Cells), Hemoglobin, Hematocrit, Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Platelet Count (Platelet)
Differential Leukocyte Count*:
- Unsegmented Neutrophil (Unsegmented)
- Lymphocyte
- Monocyte
- Eosinophil -Basophil
- Segmented Neutrophil
(Segmented) RBC Morphology, Platelet Estimate
* Performed on the control and high dose groups at the week 4 and 6 necropsies and on the mid dose group at the week 4 necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: week 4 and week 6 necropsies
- Animals fasted: Yes
- How many animals: All
- Parameters checked:
Glucose, Urea Nitrogen, Creatinine, Total Protein, Albumin, Albumin/Globulin Ratio (A/G Ratio), Globulin, Calcium, Phosphorus, Chloride, Total Bilirubin, Gamma Glutamyltransferase, Bile Acids, Serum Aspartate Aminotransferase, Serum Alanine Aminotransferase, Serum Alkaline Phosphatase, Sodium, Potassium, Total Cholesterol.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 or week 6 necropsies
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked:
Volume, Color (CLOR), Appearance (APP), Specific Gravity (SG), pH, Leukocytes (WBC), Nitrites (NIT), Protein (PRO), Glucose (GLU), Ketones (KET,) Bilirubin (BIL), Occult Blood (BLD), Urobilinogen, Microscopy of Sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly, beginning one week prior to initiation of test article exposure, and prior to the scheduled necropsy
- Dose groups that were examined: all
- Battery of functions tested: handling observations, open-field observations
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera.

ORGAN WEIGHTS: Yes
Adrenals Spleen
Brain Ovaries
Epididymides Testes
Heart Thymus Kidneys
Liver
Lungs

HISTOPATHOLOGY: Yes
Adrenals (2), Aorta, Bone with marrow (sternebrae), Brain (forebrain, midbrain, hindbrain), Eyes with optic nerve (2), Gastrointestinal tract ( Esophagus, Stomach, Duodenum, Jejunum, Ileum, Peyers' patches, Cecum, Colon, Rectum), Heart, Kidneys (2), Liver (sections of two lobes), Lungs [including bronchi, fixed by inflation with fixative (2)], Lymph node (bronchial, mesenteric, suprapharyngeal), Mammary gland (females only), Nasal cavities, Ovaries with oviducts (2), Pancreas, Parathyroids (if present), Peripheral nerve (sciatic), Pituitary, Prostate, Salivary glands [submaxillary (2)], Seminal vesicles (2), Skeletal muscle (vastus medialis), Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Testes with epididymides (2), Thymus, Thyroids, Trachea, Urinary bladder, Uterus with vagina, All gross lesions.
Statistics:
All analyses were conducted using two-tailed tests for minimum significance levels of 1% and 5% comparing the treatment groups to the vehicle control group by sex. All means were presented with standard deviations (S.D.) and the numbers of sampling units (N) used to calculate the means. All statistical tests were performed by a Digital® MicroVAX® 3400 computer with appropriate programming. Body weight, body weight change, food consumption, clinical laboratory and absolute and relative organ weight data were subjected to a one-way analysis of variance followed by Dunnett's test.
Clinical laboratory values for cell types that occur at a low incidence (monocytes, eosinophils, basophils, unsegmented neutrophils) were not subjected to statistical analyses.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY (Tables 1 through 6)
In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies.
The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level.

BODY WEIGHT AND WEIGHT GAIN (Tables 35, 36)
Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. 

FOOD CONSUMPTION (Table 37)
Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animals were similar to the control group values.

HAEMATOLOGY (Tables 38, 39, 40)
At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group mates and females, and the lymphocyte count was reduced in the females in this group.

CLINICAL CHEMISTRY (Table 41)
Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the mates in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm mates, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level.

URINALYSIS (Tables 42, 43)
Total urine volume was increased in the 1500 ppm group males and females relative to the control group values at the week 4 and week 6 evaluations; however, the increases were not statistically significant. In addition, urine volume is a highly variable parameter, and no other findings were noted in these animals that would account for this increase in volume; therefore, the changes in urine volume were not attributed to test article exposure.

NEUROBEHAVIOUR (Tables 7 through 20)
The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related findings were noted during the Functional Observational Battery evaluations

ORGAN WEIGHTS (Tables 47, 48, 49, 50)
At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the 1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy.

GROSS PATHOLOGY (Tables 44, 45, 46)
No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. 

HISTOPATHOLOGY: NON-NEOPLASTIC (Tables 51, 52, 53)
At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal nonsuppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level.
Dose descriptor:
LOAEC
Remarks:
systemic toxicity
Effect level:
1 506 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight reversible decreases in albumin and A/G ratio (females only)
Dose descriptor:
NOEC
Remarks:
systemic toxicity
Effect level:
15 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weight.
Dose descriptor:
LOAEC
Remarks:
nasal irritation
Effect level:
150 ppm (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis
Dose descriptor:
NOEC
Remarks:
nasal irritation
Effect level:
15 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: Minimal to moderate nonsuppurative inflammation, minimal to mild squamous hyperplasia and mild necrosis
Critical effects observed:
yes
Lowest effective dose / conc.:
150 ppm
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Critical effects observed:
yes
Lowest effective dose / conc.:
1 500 ppm
System:
other: liver and thymus
Organ:
liver
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified
Conclusions:
In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights-and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues.
Executive summary:

Potential subchronic toxic effects of the test article, Diethylhydroxylamine (DEHA), were evaluated in this 28-day study in rats. The test article was administered via nose-only inhalation to three groups, each comprised of 15 male and 15 female Crl:CD®BRrats, for a period of six hours per day, five days per week, for four consecutive weeks (minimum of 20 total exposures). The targeted exposure concentrations were 15, 150 and 1500 ppm. The test atmosphere concentrations were monitored by infrared absorbance and were found to be 15, 150 and 1506 ppm. A concurrent control group of identical design received only filtered air, on a comparable regimen. The animals were observed for clinical signs and effects on body weight, food consumption and clinical pathology parameters. Data from detailed physical examinations, including Functional Observational Battery data (handling and open field observations), were recorded during the pretest period and during weeks 0 through 5. After completion of exposure, 5 rats/sex/group entered an approximate two-week (nonexposure) recovery period, after which they were euthanized; necropsies were performed, and selected organs were weighed. The remaining rats in each group were euthanized immediately following the exposure period and necropsied as described above. A microscopic examination was conducted on selected tissues from all groups.

In the control, 15, 150 and 1500 ppm groups, 2, 1, 2 and 2 animals, respectively, were found dead during the study. These deaths were noted while the animals were in the exposure tube either prior to exposure, during exposure or at the time of unloading from the exposure tubes. The deaths did not occur in an exposure-related manner and were not related to exposure to the test article. All other animals survived to the scheduled necropsies. The predominant treatment-related clinical signs were dried yellow dorsal posterior and urogenital matting, lack of grooming, eye closure and hypoactivity in males and females in the 1500 ppm group, and ataxia, paleness in color, walking on tiptoes and hunched posture in the females in this group. The findings of ataxia, paleness in color, walking on tiptoes, hunched posture, eye closure and hypoactivity were transient in that they occurred only at the post-exposure observation and not prior to exposure or during the Functional Observational Battery. During the recovery period, no significant findings were noted at any exposure level. The only potential test article-related finding noted during the Functional Observational Battery evaluations (handling and open field observations) was an increase in slightly soiled or very soiled fur in the 1500 ppm group males and females during weeks 0 to 2. During the recovery period, no test article-related fmdings were noted during the Functional Observational Battery evaluations.Reductions in mean body weight gain were noted in males and females in the 1500 ppm group during week 0-1 and in males in this group throughout the remainder of the exposure period. Food consumption was reduced in the 1500 ppm group males and females during week 0-1. During the recovery period, body weights and food consumption in these animais were similar to the control group values. At the week 4 evaluation, the segmented neutrophil count was increased in the 1500 ppm group males and females, and the lymphocyte count was reduced in the females in this group. Alkaline phosphatase and phosphorous values were increased in the 1500 ppm group males and females at the week 4 evaluation. At the week 4 evaluation, albumin levels were decreased in the 1500 ppm group (both sexes) and the 150 ppm group (females only), and globulin was increased in the 1500 ppm females. These changes corresponded with decreased A/G ratios in the 1500 ppm group (both sexes) and the 150 ppm group females. A slight but statistically significant increase in alanine aminotransferase in the 1500 ppm group females (week 4) may also have been treatment-related. Bile acids were increased in the males in the 1500 ppm group at the week 4 evaluation. At the week 6 evaluation, the values for all of these parameters were similar to the control group values. (Although bile acids appeared elevated at the week 6 evaluation for 1500 ppm males, this was due to a low control value and unrelated to the test article.) Other hematology and serum chemistry values and urinalysis parameters were unaffected by exposure to the test article at any exposure level. No test article-related internal findings were noted at the necropsies of animals that died during the study or at the scheduled necropsies. At the week 4 necropsy, thymus gland weights (relative and absolute) were reduced in males and females in the1500 ppm group. Mean liver weights (absolute and relative) were increased in the 1500 ppm group females at the week 4 necropsy. Organ weights were comparable to the control group values at the week 6 (recovery) necropsy. At the necropsy of animals that died during the study, no test article-related microscopic observations were noted. At the week 4 necropsy, reversible test article-related microscopic changes consisting primarily of nonsuppurative mucosal inflammation, but also including squamous hyperplasia and necrosis in a limited number of animals, were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be local, not systemic. At the recovery necropsy, only one rat of each sex in the 1500 ppm group had minimal non suppurative mucosal inflammation in the nasal cavity. Medullary plasmacytosis was noted at an increased incidence in the iliac and popliteal lymph nodes in males in the 1500 ppm group. At the recovery necropsy, no exposure-related microscopic effects were noted in males or females at any dose level. In conclusion, toxicity was exhibited in the 1500 ppm group by clinical signs, inhibition of body weight gain and food consumption, changes in white blood cell differential counts, various serum chemistry changes, reduced thymus gland weights and increased liver weights. Medullary plasmacytosis was noted in the iliac and popliteal lymph nodes in males in the 1500 ppm group. Systemic effects in the 150 ppm group were limited to slight decreases in albumin and A/G ratio (females only). Based on data collected following a two-week nonexposure (recovery) period, all of these effects were considered to be reversible. Microscopic changes were noted in the nasal passages of male and female rats in the 150 and 1500 ppm groups; these effects were considered to be due to local irritation, not systemic toxicity, and reversible. The hematological, serum chemistry and organ weight (thymus and liver) effects in the 1500 ppm group indicate that the liver and thymus were the target organs, however, no test article related histomorphological changes were seen in these tissues. No toxicity was noted at a dose level of 15 ppm. Based on these results, exposure levels of 150 and 15 ppm were considered to be the NOAEC (no observed adverse effect concentration) and NOEC (no observed effect concentration), respectively, for systemic toxicity and the exposure level of 15 ppm was considered to be the NOEC for nasal irritation, under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
54.6 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP guideline study

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

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted according to REGULATION (EC) No 1272-2008.