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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Identification: Sec-Butyl aminoethanol
- CAS Reg. no: 35265-04-4
- Description: Colourless to yellow liquid
- Batch Number: A2053H010101
- Purity: 99.76%
- Expiry Date (Retest Date): 05-Jan-2013
- Storage Conditions: At room temperature (range of 20 ± 5 °C), light protected


Test animals

Species:
rat
Strain:
other: RccHan: WIST(SPF)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at delivery: 8 weeks
- Weight at Acclimatization:
Males: 211.5 to 240.9 g (± 7%)
Females: 152.4 to 170.6 g (± 6%)
- Fasting period before study: no
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and sterilized standard softwood bedding
- Diet: Pelleted standard Harlan Teklad 2914C ad libitum
- Water: Community tap-water ad libitum
- Acclimation period: Eight days under test conditions after health examination. Animals were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure:
other: Inhalation by nose-only, flow-past exposure
Vehicle:
clean air
Remarks on MMAD:
MMAD / GSD: Not feasible for a vapor.
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Inhalation Exposure System: Inhalation exposure was performed using a flow-past, nose-only exposure system with stainless steel tubings. The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. The flow of air at each tube was 1.0 L/min, which is sufficient to minimize re-breathing of the test atmosphere as it is more than twice the respiratory minute volume of rats
- Test Atmosphere Generation: Sec-Butyl aminoethanol was pumped into a glass flask. Compressed dried air (approximately 60 L/min) was supplied into the glass flask through a metal nebulization tube. The glass flask was kept within a thermal regulating device set to maintain a temperature in the glass flasks between approximately 90 – 110 °C to facilitate the process of vaporization. The vapor was pumped at the exit of the particle filter. Furthermore, the vapor of the test item of the low, mid and high dose group was achieved by serial dilution with compressed, filtered, dry air of the higher vapor concentration using gas pumps. The concentration of the test item administered to the animal was controlled by regulating the flow of vapor into the inhalation chamber. The generated vapor was diluted as necessary with compressed air to achieve the concentrations required for this study.

TEST ATMOSPHERE
- Exposure System Monitoring
The vapor concentration, relative humidity, temperature and oxygen concentration were measured on test atmosphere samples taken at a representative exposure port.
- Determination of the Nominal Vapor Concentration
Nominal concentration for the initially generated vapor was measured by weighing the syringe containing the test item solution before and after each exposure to determine the quantity of test item used. The weight of test item used was then divided by the total airflow volume to give the nominal concentration. These data were used only for the purpose of monitoring the performance of the generation system.
- Gravimetric Determination of Vapor Concentrations and Determination of Particle Size Distribution
Not feasible for a vapor. In addition, as a vapor the test item was considered to be respirable.
- Chemical Determination of Vapor Concentrations
To determine the concentration of the test item in the generated vapor chemical analyses of test atmosphere samples was performed once, twice or three times daily for groups 2, 3 and 4, respectively. Test atmosphere samples were collected in solvent traps with 100 ml Acetonitrile as solvent per trap. Sampling flow was 1 L/min per exposure port. The duration of sampling was between 300 to 330 min for group 2, 120 to 150 min for group 3 and 78 to 95 min for group 4. For each exposure one, two or three samples were collected for groups 2 to 4, respectively, and were transferred into labeled appropriate vials, forwarded on cool packs to the scientist responsible for formulation analysis and stored at 5 ± 3 °C until analysis or were analyzed directly. They were analyzed for the test item by Harlan Laboratories Ltd., Zelgliweg 1, 4452 Itingen / Switzerland using a GC method.
- Oxygen Concentration
The oxygen concentration in the chamber was measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure. The oxygen concentration was maintained above 19% during the exposure period.
- Relative Humidity / Temperature
The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure.
- Airflow Rate
The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters. The actual airflow rate was monitored hourly during each exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
see before
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
6 hours/day, 5 days/week at approximately 24-hour intervals.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5, 20 and 80 mg/m3
Basis:
other: target conc.
Remarks:
Doses / Concentrations:
5.979 ± 1.641, 21.15 ± 2.657 and 90.48 ± 7.770 mg/m3
Basis:
analytical conc.
No. of animals per sex per dose:
Group 1: 10 males and 10 females
Group 2: 5 males and 5 females
Group 3: 5 males and 5 females
Group 4: 10 males and 10 females
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: Dosages were selected based on data from the 5-Day dose range finding study.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily during treatment, once daily during acclimatization and recovery.
- Cage side observations checked in table [No.?] were included.

CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily during treatment (after exposure) and once weekly during acclimatization
and recovery.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly (each individual animal) during acclimatization and recovery and twice weekly during treatment.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly (per cage) during acclimatization, treatment and recovery.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Acclimatization and week 4 of treatment
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: after 4 Weeks
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- The following hematology parameters were determined:
Complete Blood Cell Count
Erythrocyte count
Hemoglobin
Hematocrit
Mean corpuscular volume
Red cell volume distribution width
Mean corpuscular hemoglobin
Mean corpuscular hemoglobin concentration
Hemoglobin concentration distribution width
Reticulocyte count
Reticulocyte maturity index (low, medium,
high fluorescence)
Leukocyte count, total
Differential leukocyte count:
Neutrophils
Eosinophils
Basophils
Lymphocytes
Monocytes
Large unstained cells
Platelet count
Coagulation
Prothrombin time (= Thromboplastin time)
Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: after 4 Weeks
- Animals fasted: Yes
- How many animals: all
- The following clinical biochemistry parameters were determined:
Glucose
Urea
Creatinine
Bilirubin, total
Cholesterol, total
Triglycerides
Phospholipids
Aspartate aminotransferase
Alanine aminotransferase
Lactate dehydrogenase
Alkaline phosphatase
Gamma-glutamyl-transferase
Creatine kinase
Sodium
Potassium
Chloride
Calcium
Phosphorus
Protein, total
Albumin
Globulin
Albumin/Globulin ratio

URINALYSIS: Yes / No / No data
- Time schedule for collection of urine: after 4 Weeks
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- The following urine parameters were determined:
Physical Examination:
Urine volume (16 hour)
Specific gravity (relative density)
Color
Appearance
Chemical Examination:
pH value
Nitrite
Protein
Glucose
Ketones
Urobilinogen
Bilirubin
Erythrocytes
Leukocytes

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table below)

ORGAN WEIGHTS: Yes (see table below)

HISTOPATHOLOGY: Yes (see table below)
Statistics:
The following statistical methods were used to analyze body weight, ophthalmoscopic examinations, macroscopic findings, organ weights and ratios, as well as clinical laboratory data:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
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
All animals survived the scheduled treatment and recovery periods.
No clinical signs were observed throughout the scheduled treatment and recovery period.

BODY WEIGHT AND WEIGHT GAIN
Slight, but not statistically significant, reductions of the body weight and body weight gain were recorded in the male animals of group 4 from week 2 until the end of the treatment period when compared to the animals of the control group. Normal body weight development was observed in these animals during the recovery period.
Body weight and body weight gain were not affected in the female animals.
Body weight loss or stagnation of the body weight gain during the first few days of inhalation studies is not unusual and is due to the restraining of the anima s in the tube during the nose-only exposure procedure.

FOOD CONSUMPTION
No effect on food consumption was observed in the animals of all groups throughout the treatment and recovery period.

OPHTHALMOSCOPIC EXAMINATION
As ophthalmoscopic findings noted at the end of the treatment period were already noted during acclimatization period they were considered not to be related to the treatment with the test item.

HAEMATOLOGY
There were no test item-related effects in hematology in males and females of all treated groups when compared to the control group.
Some inter-group variations occasionally achieved statistical significance but did not show a dose-relationship, were within the historical control range or were considered to be of normal biological variation. Therefore they were considered not to be related to treatment with the test item and are not discussed further.

CLINICAL CHEMISTRY
No effects on clinical biochemistry parameters were observed in males and females of the treated groups when compared to the control group.
Some inter-group variations occasionally achieved statistical significance but did not show a dose-relationship, were within the historical control range or were considered to be of normal biological variation and were therefore considered not to be related to treatment with the test item.

URINALYSIS
Urinalysis parameters remained unaffected by the treatment with the test item in males and females when compared to the control group.

ORGAN WEIGHTS
Organ weights of the animals treated with the test item were not different from those of the control group.

GROSS PATHOLOGY
There were no macroscopic findings during necropsy that were considered to be related to treatment with the test item.
All findings were considered to be incidental findings commonly noted in rats of this strain and age and were therefore considered not to be related to treatment with the test item.

HISTOPATHOLOGY: NON-NEOPLASTIC
Minimal focal olfactory mucosa epithelial degeneration was observed at the dorsal meatus of the nasal cavity level 2 in only one male treated with 80 µg Sec-Butyl aminoethanol 2-[(1-methylpropyl)amino]ethanol /L air (group 4) after the end of the treatment period (allocation A). A minimal focal change consisting of regenerative epithelium of the nasal cavity level 3 was noticed in one male and one female of the high dose group after the end of the recovery period (allocation B). The lesions were found either on the septum or at a turbinate tip.
The remainder of findings recorded was within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels

open allclose all
Dose descriptor:
NOAEC
Remarks:
nasal irritation
Effect level:
21 mg/m³ air (analytical)
Sex:
male/female
Dose descriptor:
NOAEC
Remarks:
systemic toxicity
Effect level:
> 90 mg/m³ air (analytical)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table1      Incidence and Mean Severity of Main Findings in Nasal Cavities

Main Study Animals (Allocation A) Nasal Cavity Level 2

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

(5) M

(5) F

Epithelial Degeneration

0

0

0

0

0

0

1/1.0

0

Recovery Animals (Allocation B) Nasal Cavity Level 3

Finding / Groups

1

2

3

4

Total Affected /
Mean Severity

(5) M

(5) F

0

0

0

0

(5) M

(5) F

Epithelial Degeneration, Regenerative

0

0

-

-

-

-

1/1.0

1/1.0

 

Applicant's summary and conclusion

Conclusions:
Daily exposure of Wistar rats to target vapor concentrations of 5, 20 and 80 µg/L Sec-Butyl aminoethanol over a period of 5 days/week for 4 consecutive weeks resulted in slight and transient effects on body weight and in minor primary changes in the nasal cavities.
Minimal effects on body weight were observed in the males of the high dose group, only, and were not present after the end of the recovery period.

Test item-related histopathologic findings were limited to nasal cavity levels 2 and 3 and consisted of minimal focal olfactory mucosa epithelial degeneration at the dorsal meatus in one high dose male after 4 weeks of treatment and of a minimal focal change consisting of regenerative epithelium in one male and female, each, of the high dose group after 4 weeks of recovery. The lesions were found either on the septum or at a turbinate tip. In accordance to these findings the nasal cavities were considered to be the target organ for the exposure to Sec-Butyl aminoethanol
There were no further findings related to treatment with Sec-Butyl aminoethanol which were considered to be of toxicological relevance.
Based on the histopathological results in this study the mid concentration of 20 µg Sec-Butyl aminoethanol /L air was considered to be the No-Observed Adverse Effect Concentration (NOAEC) for the nasal irritation. The high concentration of 80 µg Sec-Butyl aminoethanol /L air was considered to be the NOAEC for the systemic toxicity.
Executive summary:

In a 4-week inhalation toxicity study Sec-Butyl aminoethanol was administered 6 hours daily to rats by nose-only, flow-past inhalation exposure for a period of 5 days/week for 4 consecutive weeks. The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 4-week treatment free recovery period following the treatment period. The study comprised 5 male and 5 female Wistar rats in each group and additionally 5 male and 5 female rats in groups 1 and 4 for recovery. The rats of the control group 1 were exposed to air. Groups 2 to 4 were exposed to target concentrations of 5, 20 and 80 μg/L Sec-Butyl aminoethanol respectively. Throughout the study all animals were observed for viability and mortality, clinical signs, body weights, food consumption, ophthalmoscopy, clinical laboratory investigations, organ weights and macroscopic findings were recorded. A histopathologic examination was also performed. The study was performed in accordance with the procedures indicated by the OECD Guideline for the Testing of Chemicals, Number 412, September 7, 2009 "Subacute Inhalation Toxicity: 28-Day Study" and in compliance with Good Laboratory Practice. Exposure to chemically determined vapor concentrations of 5.98, 21.2 and 90.5 μg/L Sec-Butyl aminoethanol were achieved in groups 2 to 4, respectively, and were slight to moderately above the respective targets. Temperature, relative humidity and oxygen concentration during exposure were considered to be suitable for this type of study. All animals survived and no clinical signs were observed during the scheduled treatment and recovery period. Slight, but not statistically significant, reductions of the body weight and body weight gain were recorded in the males of group 4 from week 2 until the end of the treatment period and returned to normal during the recovery period. Body weight in females was not affected. There was no effect on food consumption. No test item-related findings were recorded in ophthalmoscopy. Parameters in hematology, clinical biochemistry and urinalysis remained unaffected. There was no effect on organ weight. No test item-related macroscopic findings were recorded during necropsy. Microscopic findings consisted of minimal focal olfactory mucosa epithelial degeneration at the dorsal meatus of the nasal cavity level 2 in one high dose male (allocation A) and of a minimal focal change consisting of regenerative epithelium of the nasal cavity level 3 in one male and one female of the high dose group (allocation B). Lesions were found either on the septum or at a turbinate tip.

In accordance to the findings above the nasal cavities were considered to be the target organ for the exposure to Sec-Butyl aminoethanol. Based on the histopathological results in this study the mid concentration of 21 μg Sec-Butyl

aminoethanol /L air was considered to be the No-Observed Adverse Effect Concentration (NOAEC) for the nasal irritation. The high concentration of 90 μg Sec-Butyl aminoethanol /L air was considered to be the NOAEC for the systemic toxicity.