O-ethylhydroxylamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions (no data on substance purity, no neurobehavioral evaluations)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1987

Report date:

1987

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

No neurobehavioural evaluations

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

O-ethylhydroxylamine

EC Number:

402-030-3

EC Name:

O-ethylhydroxylamine

Cas Number:

624-86-2

Molecular formula:

C2 H7 N O

IUPAC Name:

O-ethylhydroxylamine

Details on test material:

- Source: Imperial Chemical Industries PLC
- CTL reference number: Y05460/002/001
- Name of test substance as cited in report: Substance H109360
- Identification of test sample as cited in report: 50% (w/v) aqueous solution (46.4% Substance H109360 and approximately 5% ethanol)
- Characterization of test substance: volatile organic amine (colourless liquid)
- Lot #: EFI-100
- Analytical purity: not specified
- Storage: at room temperature
- Impurities: Ethanol was known to be present in the test substance in concentrations of upto 5% but was not analysed as it was considered that it would not contribute to any toxicity seen.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Name as cited in report: Alpk:AP (Wistar derived) albino rats
- Source: from the colony maintained at Alderley Park, Cheshire, UK
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: males 233-269g ; females 200-235g
- Fasting period before study: not specified
- Housing: 5/cage/sex in long term exposure chambers
- Diet: Pelleted Porton Combined (PC) diet, provided ad libitum
- Water; provided ad libitum
- Acclimation period: 9 days
The known contaminants found in food and water were considered not to be present in sufficient concentration as to have had an influence on the outcome of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C):19-24
- Humidity (%): 35-67
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The chambers (Doe and Tinston, 1981) were of stainless steel and had an internal volume of 3.4m3
- Method of holding animals in test chamber: in long term exposure chambers
- Method of conditioning air: not specified
- System of generating aerosols: concentric jet atomisers
- Temperature, humidity in air chamber: 20-26 (nominal), 40-46% (nominal)
- Air flow rate: 300 l/min (control, 50, 100 ppm) or 400-450 1/min (5 ppm)
- Treatment of exhaust air: not specified


TEST ATMOSPHERE
- Brief description of analytical method used: gas chromatography with a flame ionization detector. This was set at 2ppm based on a 2 ml sample volume
- Samples taken from breathing zone: yes


Doe and Tinston, 1981 Novel Chambers for Long Term Inhalation Studies. Proceedings of the Inhalation and Technology Symposium, Kalamazoo, Michigan 1980. Ed Leong BJK Ann Arbor Science.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analysed atmospheric concentrations of organic amine indicated that these exceeded the target concentrations, particularly at the lowest dose level (approx. 10 ppm vs. 5 ppm targeted concentration). This was attributable to analytical problems at the lower concentrations generated, which resulted in an underestimation of the true atmospheric concentrations. Nonetheless, the concentrations remained acceptably stable throughout the study. The similarity between the nominal and analysed concentrations demonstrates that Substance H109360 was almost completely volatilised and that there were only minor losses within the generation and exposure systems.

Duration of treatment / exposure:

28 consecutive days

Frequency of treatment:

6 h/d

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 5, 50, 100 ppm
Basis:
other: Target concentrations

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 10.1, 61.3, 123 ppm (0, 0.025, 0.153 and 0.306 mg/l) (calculated from the weight loss of the formulation)
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
0, 10.8, 59, 105 ppm (0.03, 0.15, 0.26 mg/l) (chemical analysis)
Basis:
analytical conc.

No. of animals per sex per dose:

Control: 10 (5 for satellite)
5 ppm: 5
50 ppm: 5
100 ppm: 10 (5 for satellite group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the findings of a 4-hour acute inhalation study (Hext and Leah, 1981), and from preliminary trials to the present study.
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: test for reversibility of eventual effects and/or the onset of late effects
- Post-exposure recovery period in satellite groups: 14 days
- Doses included in the satellite: 0 and 100 ppm
- No. of animals in satellites: 5/sex/dose

Hext P M and Leah A M (1987). Substance H109360 : 4 Hour Acute Inhalation Toxicity Study in the Rat. CTL Report No : CTL/P/1198

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to, during (approximately every 30 minutes) and following each exposure and daily during the maintenance period

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: following exposure on days 1, 2, 3, 5, 8, 15, 22, 29, 36 and 43
- Parameters checked: Condition of fur, respiration, motor activity, skin colour, salivation, faeces, righting reflex, condition of eyes, corneal reflex, pinna reflex, foot withdrawal reflex, response to sound

BODY WEIGHT: Yes
- Time schedule for examinations: on day -1, prior to exposure on days 1, 2, 3, 5, 8, 15 and 22, and on days 29, 36 and 43

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following exposure on day 28, and on day 42
- Dose groups that were examined: control and 100 ppm dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination on days 29 and 43
- Anaesthetic used for blood collection: Yes (halothane)
- Animals fasted: not specified
- How many animals: all animals of all groups
- Parameters checked: Platetet count, red cell count and total white cell count, haemoglobin and haematocrit, Mean cell volume, mean cell haemoglobin and mean cell haemoglobin concentration, a peripheral blood film was prepared, a differential white cell count was performed on this film and in addition, the morphological appearance of red cells was examined. In addition kaolin-cephalin and prothrombin times were measured. Bone marrow smears were also taken but were not examined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination on days 29 and 43
- Animals fasted: not specified
- How many animals: all animals
- Parameters checked: Plasma urea, glucose, albumin, total protein and cholesterol, Ppasma alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and triglycerides

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

BLOOD PRESSURE MONITORING
- Rationale: During the trial exposures, there were pathological changes in the spleen which were possibly due to blood pressure elevation. Therefore the blood pressure of the animals was tested
- Dose groups examined: control and 100 ppm dose groups
- Time schedule: following exposure on days 8, 14, 21 and 28 and on day 42 (recovery group)
- Method: Systolic blood pressure was measured indirectly in conscious rats using an occlusive cuff (on the tail) connected to an REL blood pressure monitor

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- Dose groups that were examined: all animals
- Parameters checked: particular attention being given to the abdominal and thoracic viscera

ORGAN WEIGHTS: Yes
- Doses that were examined: all doses
- Lungs (with trachea attached but larynx removed), heart, liver, kidneys, spleen, adrenal glands and testes were weighed. Paired organs were weighed together

HISTOPATHOLOGY: Yes
- Doses that were examined: all animals of the control and 100 ppm dose group
- Parameters checked: the head, for nasal turbinates (after perfusion with formol saline), brain, lungs inflated with formol saline, larynx, trachea, heart, liver, spleen(examined for all dose groups), kidneys, adrenal glands, prostate gland, seminal vesicles or uterus, cervix and ovaries and any abnormal tissues, testes, epididymides, eyes

Statistics:

Where appropriate, test and control data were compared statistically using a two-sided Student's t-test

Results and discussion

Results of examinations

Details on results:

MORTALITY
- There were no mortalities in any group

CLINICAL SIGNS
Exposure to concentrations of up to 105 ppm of the organic amine did not result in severe clinical toxicity. Clinical abnormalities recorded were indicative of a mild, non-specific response to exposure with some indications of respiratory tract irritation in a few animals exposed to 105 ppm. Respiratory tract irritation was miirored by minor histopathological changes in the respiratory tract. This may account for the small increases seen in some lung-bodyweight ratios at the end of the exposure and probably reflects the general irritant capacity of amines.

- During Exposures : No abnormalities were seen at any time in animals exposed to 10.8 ppm or the control group. Some of those exposed to 59 ppm had slight piloerection during most of the 28-day exposure period. All those exposed to 105 ppm had slight piloerection on most days and some were slightly hunched.
- Immediately after exposure: No significant abnormalities were seen at any time in the control group or those exposed to 10.8 ppm. A few animals exposed to 59 ppm had hunched posture and piloerection throughout the 28-day exposure period. Hunched posture and piloerection were also seen throughout exposure in those exposed to 105 ppm, but the incidence was greater in the higher dose group and in addition several animals were making abnornal respiratory noises.
- Post exposure period (days 29-43 ): There were no significant abnormalities in the control group or prior to post mortem examination on day 29 in those exposed to 10.8 or 59 ppm. The only significant abnormality in those exposed to 105 ppm was piloerection which persisted until day 36 in four animals.

BODY WEIGHT AND WEIGHT GAIN
Both sexes exposed to 105 ppm lost weight slightly the day following the first exposure and males exposed to 59 ppm gained less weight than controls at this time. Thereafter, the day-to-day weight gain of all groups was similar to controls, although the overall weight gain of those exposed to 105 ppm remained lower than controls due to their initial weight loss. These bodyweight effects were reflected by small reductions in food consumption during the exposure phase of the study.

FOOD CONSUMPTION
- The food consumption of animals exposed to 105 ppm was slightly lower than controls until day 8. Other groups were unaffected .

OPHTHALMOSCOPIC EXAMINATION
- No treatment related changes were found.

HAEMATOLOGY
Treatment period: There was a dose-related decrease in the haemoglobin and related parameters (haematocrit and red cell count) on day 29 in both sexes, with an associated dose-related increase in mean cell volume and mean cell haemoglobin. The magnitude of the effect on the mean cell volume was greater in females than in males but the effect was evident in all rats exposed to 10.8 ppm. Target cells were seen in most rats exposed to 59 or 105 ppm.
- Haemoglobin: female: 98%, 89% (p<0.05) and 88% (p< 0.01) of control value; male: 95%, 94% and 89% (p<0.01) of control value in 10.8, 59 and 105 ppm, respectively
- Haematocrit: female: 102%, 95% and 93% of control value; male: 99%, 96% and 92% (p< 0.05) of control value in 10.8, 59 and 105 ppm, respectively
- Red blood cell count: female 91%, 74% (p<0.01) and 67% (p<0.01) of control value; male: 91.5% (p<0.05), 77% (p<0.01)and 68% (p< 0.01) of control value in 10.8, 59 and 105 ppm, respectively
- MCV: female 108%, 124% (p<0.01) and 125% of control value; male: 114% (p<0.05), 131% (p<0.01)and 140% (p< 0.01) of control value in 10.8, 59 and 105 ppm, respectively
- MCH: female 108% (p<0.05), 121% (p<0.01) and 131% (p<0.01) of control value; male: 104%, 120% (p<0.01)and 130% (p< 0.01) of control value in 10.8, 59 and 105 ppm, respectively

Post treatment period: On day 43, the haemoglobin and related parameters for the test animals were increased in both sexes compared with the controls. The mean cell volume of test animals was also increased when compared with controls although the effect was less pronounced than on day 29.

CLINICAL CHEMISTRY
- Treatment period: There was no evidence of overt toxicity. Plasma ALT activities were slightly reduced but with statistical significance in females exposed to 59 ppm (62% of control value; p< 0.05) and both sexes exposed to 105 ppm (in each sex; approx. 61% of control value; p< 0.01). Plasma AST activities were also slightly reduced (72% of control value; p< 0.05) in males exposed to 105 ppm. There was a slight reduction in plasma triglycerides in females exposed to 59 ppm (71% of control value) and males (66% of control value) exposed to 105 ppm although this was only of statistical significance (p< 0.05) in the females. Total protein was slightly reduced (92% of control value) in females exposed to 105 ppm. There were other minor changes, which although of statistical significance, are not considered to be of toxicological significance.
- Post exposure period: There were no marked effects, and the changes seen on day 29 were no longer apparent.

ORGAN WEIGHTS
- Treatment period: There was a large and dose-related increase in spleen weight and spleen:bodyweight ratio in test animals compared with controls. The percentage increase ranged from 24% to 527%. There was an increase in the heart weight and heart:bodyweight ratio of males exposed to 105 ppm, but this was due to one animal. There was also an increase in the heart:bodyweight ratio of females exposed to 59 or 105 ppm compared with controls, although the percentage increase was small (< 10%). There was a small (< 10%) but dose-related increase in the lung:bodyweight ratio of females in all test group. The increase in heart weights may have been due to the anaemia causing reduced oxygen capacity with cardiac hypertrophy occurring as a compensating mechanism.
- Post exposure period: After non treatment recovery period, there was a slight increase in the liver weight and liver bodyweigt ratios of females exposed to 105 ppm. There was an increase of approximately 40% in the spleen weight and spleen:bodyweight ratio of both sexes exposed to 105 ppm.

GROSS PATHOLOGY
- Treatment period: No treatment-related abnormalities were found in controls or in females exposed to 10.8 ppm. All animals (both sexes) exposed to 59 or 105 ppm and one male exposed to 10.8 ppm had enlarged spleens, some of which were dark red and/or misshapen
- Post exposure period: One female exposed to 105 ppm had a spleen with a roughened surface

HISTOPATHOLOGY:
TREATMENT PERIOD
In the spleen there was extramedullary haemopoiesis (slight to moderate), congestion (minimal to slight), haemosiderosis (minimal to slight) and focal capsular fibrosis (minimal). The incidence and severity of these changes increased with increasing dose-level and correlated with increased spleen weights. The spleens of control animals were normal.
Extramedullary haemopoiesis (spleen)
- males: control (0/5), 10.8 ppm (5/5), 59 ppm (5/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (5/5), 59 ppm (5/5), 105 ppm (5/5)

Congestion (spleen)
- males: control (0/5), 10.8 ppm (5/5), 59 ppm (5/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (5/5), 59 ppm (5/5), 105 ppm (5/5)

Capsular fibrosis (spleen)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (1/5), 105 ppm (2/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (3/5), 105 ppm (2/5)

Haemosiderosis (spleen)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (5/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (5/5), 105 ppm (5/5)

There was minimal extramedullary haemopoiesis and a few iron-containing Kupffer cells in the livers of animals exposed to 105 ppm. No changes were observed in the control livers.
Extramedullary haemopoiesis (liver)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)

Pigmented Kuppfer cells (liver: minimal to slight pigmentation)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)

Inflammatory changes were seen in the anterior segments of the nasal passages of both exposed and control animal.
- Squamous metaplasia (males): control (1/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (4/5)
- Squamous metaplasia (females): control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (3/5)
- Rhinitis (males): control (2/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)
- Rhinitis (females): control (2/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)
.
POST EXPOSURE PERIOD
Increased extramedullary haemoposiesis seen in the spleen at the termination of the treatment period was no longer increased compared to the controls indicating that the effects had been reversed.
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (0/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (0/5)

Haemosiderin deposits in the spleens of animals exposed to 105 ppm had increased after the recovery period, and were moderate in severity. This change was not present in controls.
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)

There was some congestion in the spleens of animals of both sexes at 105 ppm, but the congestion was not as severe as that seen directly after exposure. No congestion was seen in controls.
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (4/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (5/5)

Minimal capsular fibrosis was present in male animal of the 105 ppm dose group.

The incidence of extramedullary haemopoeisis in the liver was reduced after the recovery period. There was minimal extramedullary haemopoiesis in two males exposed to 105 ppm and a few pigmented Kupffer cells were still apparent in some animals exposed to 105 ppm.
Extramedullary haemopoiesis (liver)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (2/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (0/5)

Pigmented Kuppfer cells (liver: minimal pigmentation)
- males: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (4/5)
- females: control (0/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (1/5)

There was evidence of inflammatory change in the nasal passages at this time period but the incidence was similar in test and control animals indicating that the change was not treatment related. Changes seen in other tissues were considered to be incidental to treatment.
- Squamous metaplasia (males): control (5/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (3/5)
- Squamous metaplasia (females): control (3/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (4/5)
- Rhinitis (males): control (3/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (4/5)
- Rhinitis (females): control (2/5), 10.8 ppm (0/5), 59 ppm (0/5), 105 ppm (0/5)

BLOOD PRESSURE MONITORING
- There were no treatment-related effects on systolic blood pressure.

Effect levels

Dose descriptor:

LOAEC

Remarks:

(28 days)

Effect level:

0.03 mg/L air (analytical)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the repeated inhalation exposure to low concentrations of Substance H109360 induced dose-related hematological changes indicative of macrocytic anemia, as well as splenic congestion seen in all groups. These results lead to the conclusion that Substance H109360 may affect certain organs.