2-ethylhexylamine

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Version / remarks:

7 September 2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.29 (Sub-Chronic Inhalation Toxicity:90-Day Study)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3465 (90-Day Inhalation Toxicity)

Version / remarks:

Agust 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-ethylhexylamine

EC Number:

203-233-8

EC Name:

2-ethylhexylamine

Cas Number:

104-75-6

Molecular formula:

C8H19N

IUPAC Name:

2-ethylhexylamine

Specific details on test material used for the study:

- BAtch no.: 000STD77L0

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rats were selected since this rodent species is recommended in the respective test guidelines. Wistar rats were selected since there is extensive experience available in the laboratory with this strain of rats.
Wistar rats, Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; Sandhofer Weg 7, 97633 Sulzfeld
- Housing: together (5 animals per cage) in Typ 2000P ca. 2065 cm2 (polysulfone cages)
- Diet: ad libitum (mouse/rat laboratory diet “GLP”, 12 mm pellets)
- Water: ad libitum
- Acclimation period: The animals were delivered and subjected immediately to the acclimatization period in which they were adapted to the surroundings.

DETAILS OF FOOD AND WATER QUALITY: The food used in the study was assayed for chemical as well as for microbiological contaminants. The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal, Germany and the Environmental Analytics Water/Steam Monitoring of BASF SE as well as for the presence of microorganisms by a contract laboratory

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 -70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF THE INHALATION ATMOSPHERES

Generator systems:
Continuous infusion pumps PHD Ultra (Harvard Apparatus, Inc., Holliston, Massachusetts, U.S.A.)
Two-component atomizers (stainless steel, Model 970; Düsen-Schlick GmbH, Untersiemau/Coburg, Germany)
Glass mixing stages (BASF SE, Ludwigshafen, Germany)
Glass dilution tube (BASF SE, Ludwigshafen, Germany) only test group 1
Glass cyclonic separators (BASF SE, Ludwigshafen, Germany)

Generation procedure:
For each concentration, a respective constant amount of the test substance was supplied to a two component atomizer by means of a metering pump and sprayed with compressed air into a glass mixing stage. The so generated aerosol were mixed with clean conditioned air and passed via a cyclonic separator into the inhalation system. The initially generated aerosol droplets evaporated immediately due to the high vapor pressure, vapor atmosphere were passed into the exposure system.

ANALYSES
Calculation of nominal concentrations:
The nominal concentration was calculated from the study means of the test pump rates and the supply air flows used during exposure to generate the respective concentrations.

Analytical determination of concentrations:
From 20 Jan 2016 to 11 Feb 2016 the concentration was determined by offline gas chromatography (GC) analyses in test groups 1 - 3. Absorption samples were draw three times per concentration and exposure day in test groups 2 and 3, and once daily for test group 1. During this time in test group 0 two samples were draw on different days.
From 11 Feb 2016 onward, atmospheric concentrations of test groups 2 and 3 were measured by online IR spectrometer. The target concentration of test group 1 is below the detection limit of IR spectrometer and was continued being measured by offline GC analysis of daily absorption samples through the whole study period.
The off-line GC analyses of the inhalation atmospheres were performed in the Laboratory Analytical Chemistry of Experimental Toxicology and Ecology of BASF SE, in compliance with the Principles of Good Laboratory Practice.
The online infra-red spectrometry analyses were carried out as a separate study at the Competence Center Analytics, BASF SE, Ludwigshafen, Germany under the responsibility of the Study Director of this facility. The Study was in compliance with the Principles of Good Laboratory Practice.
In addition, the constancy of concentration in the test group 1 were surveyed continuously with a total hydrocarbon analyzer (FID, Testa). Background level were measured by FID in test group 0 from 04 Feb 2016 onward. The FID was calibrated with certificated test gas propane, and matched with GC analysis during the technical trial. The online FID analysis of the concentrations of the inhalation atmospheres will be performed in the Inhalation Laboratory of Experimental Toxicology and Ecology of BASF SE.

Real time monitoring of constancy of concentrations:
Total hydrocarbon analyzers (Testa 123) were used to continuously monitor the constancy of concentrations of test substance vapors in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices. The measurements were recorded using line recorders and transferred to the automated measuring system.


EXPOSURE SYSTEM; EXPOSURE OF THE ANIMALS

Conditioned air:
The central air conditioning system provides cold air of about 15°C. This cold air will passes through an activated charcoal filter, is adjusted to room temperature of 20 to 24°C and passes through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.

Exhaust air:
The exhaust air was filtered and conducted into the exhaust air of the building.

Nose-only exposure systems
The inhalation atmosphere was maintained inside aerodynamic exposure systems (INA 60, volume V  90 L, BASF SE) consisting of a cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol.
The exposure systems were located in exhaust hoods in an air conditioned room.

Exposures
The nose-only exposure technique was preferably selected for this inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult. The test substance may be irritant to the skin.
Furthermore, by using the dynamic mode of operation with a low volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of the final target concentration) is with about 4 min shorter than that of whole body chambers with a higher chamber volume.
A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures.
The animals did not have access to water or feed during the exposure

Measurements of the exposure conditions:
Principles of recording with the automated measuring system: Each parameter was measured at appropriate measuring points using suitable measuring equipment (sensors, orifice plates etc.). The measurements were standardized (0-20 or 4-20 mA) and transferred to instrumentation consoles. There, the measured values were displayed in an analogous way (where this is provided for) and some were used as actual value for regulating the specific parameter.

In addition, the measured values were scanned every 10 seconds, converted from analog to digital, transferred to a personal computer, displayed on its screen, and saved on hard disk. The computer checked the arriving values against preset threshold values, displayed warnings if violations of thresholds occurred and recorded the start and the end of threshold violations for each measured parameter affected. After the end of each exposure all data gathered during this exposure were backed up on optical media.
Daily protocols were prepared from the recorded values using suitable software. The protocols include start and stop times of exposure and possible threshold violations, and daily means of each parameter. The records saved on optical media and the printed daily records are considered as raw data. Relevant disturbances were reported.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Real time surveillance of the inhalation atmospheres with scattered light photometers /total hydrocarbon analyzers /a spectrophotometer /generally proved the constancy of each concentration throughout the daily exposures. During some daily exposures oscillations/a higher variability occurred /in the low and low intermediate concentration . The reason for this behaviour could not be identified, because generation and exposure procedure was not changed allover the study. It was not reflected in the analytically determined concentrations, which integrate the variations over the sampling time. The structured oscillations in test groups 1 and 2 coincide with the pump cycles of used and thus are caused by some mechanical irregularities in the continuous infusion pumps influencing the constancy of test substance flow to the generator. They are considered of no relevance for the study.
Real time surveillance of the inhalation atmospheres with scattered light photometers showed some variations of concentration during daily exposure, reflecting the variation found for the study means.

Duration of treatment / exposure:

90 days

Frequency of treatment:

6 hours per day on 5 consecutive days per week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

5 mg/m³ air

Remarks:

corresponds to 4.9 mg/m3 (analytical)

Doses / concentrations 2

Dose / conc.:

25 mg/m³ air

Remarks:

corresponds to 25.0 mg/m3 (analytical)

Doses / concentrations 3

Dose / conc.:

125 mg/m³ air

Remarks:

corresponds to 127.7 mg/m3 (analytical)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A 14-Day range finding study was conducted. Five male animals per group were exposed nose only to three different vapour concentrations (5, 30 and 150 mg/m3) for 6 h per day, 5 days per week for 14 consecutive days. Based on the findings (degeneration/regeneration of nasal cavity level I at 150 mg/m3) the follwoing concentrations were selected for the main study: 5 mg/m3, 25, mg/m3 and 125 mg/m3.

- Rationale for selecting satellite groups: To detect reversibility of the effects, recovery group animals (5 male and 5 female animals per group) were exposed to high concentration. A concurrent control group was exposed to conditioned air. The recovery period was 4 weeks.

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days and once a day on Saturdays, Sundays and public holidays
- Cage side observations: mortality, signs of toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days

BODY WEIGHT: Yes
- Time schedule for examinations: at the start of the pre-exposure, at the start of the exposure period and then, as a rule, once a week as well as prior to gross necropsy; animals which underwent neurofunctional testing were additionally weighed on the days of examination

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Time schedule: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the start of expsoure (day -12/-4) and at the end of the study (day 85), respectively
- Dose groups that were examined: all main group animals (day -12/4); test group 0 (control group), tes group 2 and test group 4 (high concentration) (day 85)
- Before the start of the observation period (day 92) the eyes of all post-exposure group animals, and at the end of the observation period (day 118/119/120) the eyes of the animals of test group 01 (control group), test group 21 and test group 41 (high concentration) were examined, additional.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the morning
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals per test group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the morning
- Animals fasted: Yes
- How many animals: all animals per test group
- Parameters checked in table 2 and 3 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the exposure period (day 0) and on study days 42 and 84
- Dose groups that were examined: on all male and female animals
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

OTHER:
SPERM PARAMETERS
After the organ weight determination, the following parameters were determined in the right testis or right epididymis of all male animals sacrificed on schedule: sperm motility, sperm morphology, sperm head count (cauda epididymis) and sperm head count (testis)

Sperm motility examinations were carried out in a randomized sequence. Sperm head count and sperm morphology were evaluated in controls and the high dose group, only.

Sacrifice and pathology:

PATHOLOGY
Necropsy:
The animals were sacrificed under pentobarbitone anesthesia. The left and right brachial vessels were opened by deep cuts through the pectoral muscles along both sides of the rib cage. Caution was exercised to avoid destruction of the axillary lymph nodes. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights
The following weights were determined in all animals sacrificed on schedule: Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/tissue fixation
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymis, left, Esophagus, Extraorbital lacrimal gland , Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lungs, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary glands, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testis, left, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

In case of macroscopic findings in the right testis, this testis as well as the corresponding epididymis were fixed for histopathological examination and the left testis and epididymis were used for sperm parameters.

Histopathology
Fixation was followed by histotechnical processing, examination by light microscopy and assessment of findings according to the table below:
Organs
Test group 0 1 2 3
All gross lesions A2 A2 A2 A2
Adrenal glands A1 A1
Aorta A1 A1
Bone marrow (femur) A1 A1
Brain A1 A1
Cecum A1 A1
Colon A1 A1
Duodenum A1 A1
Epididymis, left A1 A1
Esophagus A1 A1
Eyes with optic nerve A1 A1
Extraorbital lacrimal gland A1 A1
Femur with knee joint A1 A1
Harderian gland A1 A1
Heart A1 A1
Ileum A1 A1
Jejunum A1 A1
Kidneys A1 A1
Larynx (3 levels)a A1 B4 B4 A1
Larynx A3 A3
Liver A1 A1
Lung A1 A1
Lymph nodes
(Tracheobronchial,
mediastinal, mesenteric) A1 A1
Mammary gland (female) A1 A1
Nasal cavity (4 levels)b A1 B4 B4
Nasal cavity level 1 A1 A1
Nalsal cavity level 2 A3 A3
Ovaries A1 A1
Pancreas A1 A1
Parathyroid glands A1 A1
Pharynx A1 B1 B1 A1
Pituitary gland A1 A1
Prostate A1 A1
Rectum A1 A1
Salivary glands
(Mandibular and
sublingual glands) A1 A1
Sciatic nerve A1 A1
Seminal vesicles A1 A1
Skeletal muscle A1 A1
Skin A1 A1
Spinal cord
(cervical, thoracic and
lumbar cord) A1 A1
Spleen A1 A1
Sternum with marrow A1 A1
Stomach
(forestomach and
glandular stomach) A1 A1
Teeth A1 A1
Testis, left A1 A1
Thymus A1 A1
Thyroid glands A1 A1
Tracheac A1 A1
Urinary bladder A1 A1
Uterus A1 A1

Abbreviations:
A = Hematoxylin and eosin (H&E) stain
B = Paraplast embedding
1 = all animals/test group
2 = all animals affected/test group
a one level does include the base of the epiglottis
b one level will include nasopharyngeal duct; the 4 levels allow adequate examination of the squamous, transitional, respiratory and olfactory epithelium, and the draining lymphatic tissue (NALT)
c one transverse section and one longitudinal section through the carina of the bifurcation of the extrapulmonary bronchi

Statistics:

Statistical analyses see table below

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

The detailed clinical observations did not reveal any abnormalities during the whole exposure period.

Mortality:

no mortality observed

Description (incidence):

No deaths were recorded throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of female animals of all test groups were not statistically significantly different from the control group 0 throughout the whole study period.
The mean body weight of the male animals of the high concentration (125 mg/m³) was significantly lower than the concurrent control group.

The lowered mean body weight changes of male animals of test group 1 and female animals of test group 2 were considered incidental due to the lack of concentration-response relationship. In male animals of group 1 from day 82 to 86 the mean body weight change was higher than the concurrent control, whereas lower body weight changes were observed in male animals of the high concentration group starting from day 19 to 23 onward. Thus, the increased mean body weight change was considered incidental.

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmologic examinations did not show any impairment of the refracting media.
Spontaneous findings such as remainders of the pupillary membrane or corneal stippling were observed in several animals of all test groups and the control group on study day -5/-6 prior to the exposure period. On study day 90/89, cornea striping was observed in test group 0 and 3 in several males and one female each.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among hematological parameters were observed.

After the third study months in males of test group 3 (125 mg/m3) relative reticulocyte counts were slightly lower compared to the study controls and also below the historical control range (males, relative reticulocytes 1.5-2.0 %). However, this was the only altered red blood cell parameter among these individuals. Therefore, this change was regarded as treatment-related but not adverse (ECETOC Technical Report No. 85, 2002).

Regarding the differential blood cell counts in males of test group 1 (5 mg/m3) relative neutrophil counts were increased and relative lymphocyte counts were decreased. In females of test groups 1 and 2 (5 and 25 mg/m3) absolute and relative eosinophil counts as well as relative large unstained cell (LUC) counts were higher compared to study controls. However, all mentioned cell counts were not dose-dependently changed. Therefore, the alterations were regarded as incidental and not treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related, adverse changes among clinical chemistry parameters were observed.

After the end of the administration period in males of test group 3 (125 mg/m3) total bilirubin values were increased and chloride levels were decreased. Bilirubin levels were marginally above the historical control range whereas the chloride mean was within this range (males: total bilirubin 0.56-2.44 µmol/L; chloride 98.8-105.9 mmol/L). Higher total bilirubin values were the only changed clinical chemistry parameter in these individuals and therefore this alteration was regarded as treatment-related and not adverse (ECETOC Technical Report No. 85, 2002).

In females of test group 3 (125 mg/m3) creatinine, total protein and albumin levels were decreased. However, all values were within historical control ranges (females: creatinine 28.4-41.3 µmol/L; total protein 63.21-68.63 g/L; albumin 35.89-43.65 g/L).

Therefore, the decreased chloride levels in males and the decreased creatinine, total protein and albumin levels in females of test group 3 (125 mg/m3) were regarded as incidental and not treatment-related.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

On the day of the performance of the Functional Observation Battery, the animals were not exposed to the test substances.
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between treated groups and controls, were without a dose-response relationship or occurred in single animals only, these observations were considered to have been incidental

Significantly higher foot strength of fore limb was observed (p ≤ 0.05) in male animals of mid concentration group (25 mg/m³). Because there were no concentration-response relationship, this finding was considered incidental. Thus, no substance-related findings were observed for this parameters.

Home cage observations, open field observations, sensorimotor tests and motor activity tests were without substance-related findings.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

ABSOLUTE ORGAN WEIGHTS
When compared with control group 0 (=100%), the mean absolute liver weights in male animals were significantly decreased (83%) and the mean absolute kidney weights in female animals were significantly increased (110%), both in test group 3 (125 mg/m³).

RELATIVE ORGAN WEIGHTS
When compared with control group 0 (=100%), the mean relative adrenal glands weights in male animals were significantly increased (119%) and the mean relative kidney weights in female animals were significantly increased (108%), both in test group 3 (125 mg/m³).

The increased mean relative weight of the adrenal glands of male animals of test group 3 was regarded as treatment-related even in absence of a histopathological correlate as both absolute (67.2 mg) and relative (0.022%) weights were above historical controls (Historical controls absolute 56.3 - 66.0 mg, relative 0.018 - 0.019%). This finding was regarded as non-adverse.
The decreased mean absolute liver weights of test group 3 male animals were regarded as secondary to the decreased terminal body weight (- 11%, not statistically significant).

The increased mean absolute (1.339 g) and relative (0.686%) kidney weights of test group 3 female animals were within historical controls (absolute 1.26 - 1.409 g, relative 0.628 - 0.7%) and they did not show a histopathological correlate. Therefore their significance is questionable and they were considered not treatment - related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related findings were observed in larynx, level I, nasal cavity, level I and II in male animals and in nasal cavity, level I of female animals.
Larynx: In male animals, epithelial alteration in level I of the larynx was seen in all test groups including in one animal in the control group.
Nasal cavity: Degeneration/regeneration of the transitional epithelium was noted in test group 3 animals of both sexes in level I (graded minimal to moderate) and in males also in level II (graded minimal).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Concerning motility of the sperms and sperm head counts in the testis and in the cauda epididymidis no treatment-related effects were observed. The incidence of abnormal sperms in the cauda epididymidis in test group 3 (125 mg/m3) was also not statistically significantly increased. The mean values was within the historical control range (percentage of abnormal sperms in the cauda epididymidis 6.0 – 7.5 %). Therefore, no treatment-related effect was observed even with this parameter

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

25 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOEC

Effect level:

125 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects were observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

125 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

larynx

nasal cavity

Any other information on results incl. tables

Relative increase of absolute weights

	Male animals			Female animals
Test group (mg/m³)	1 (5)	2 (25)	3 (125)	1 (5)	2 (25)	3 (125)
Terminal body weight	93%	94%	89%
Kidney				106%	104%	110%**
Liver	88%	94%	83%**

* : p <= 0.05, **: p <= 0.01

Relative increase of relative weights

	Male animals			Female animals
Test group (mg/m³)	1 (5)	2 (25)	3 (125)	1 (5)	2 (25)	3 (125)
Adrenal glands	103%	104%	119%**
Kidneys				101%	101%	108%**

* : p <= 0.05, **: p <= 0.01

 

Histological findings in larynx, level I

In male animals, epithelial alteration in level I of the larynx was seen in all test groups including in one animal in the control group.

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10
Epithelial alteration	1	4	4	5
Grade 1	1	3	2	5
Grade 2		1	2

 

Histological findings in nasal cavity, level I

Degeneration/regeneration of the transitional epithelium was noted in test group 3 animals of both sexes in level I and in males also in level II.

	Male animals				Female animals
Test group (mg/m³)	0 (0)	1 (5)	2 (25)	3 (125)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10	10			10
Degeneration/regeneration, transitional epithelium				9				9
Grade 1				3				2
Grade 2				4				4
Grade 3				2				3

 

Histological findings in nasal cavity, level II

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (25)	3 (125)
No. of animals	10	10	10	10
Degeneration/regeneration, transitional epithelium	1	4	4	5
Grade 1				5

 

Applicant's summary and conclusion

Conclusions:

Inhalation exposure of 5, 25 and 125 mg/m³ 2-Ethylhexylamine for 90 days (65 exposures) caused clearly treatment-related adverse histological changes in the nasal cavity levels I and II. Moreover, slight but significantly lower body weight and body weight gain was observed in male animals of the high concentration group (125 mg/m³). No systemic effect were observed. A No Observed Adverse Effect Concentration (NOAEC) was 25 mg/m³ under the current study conditions.

Executive summary:

To evaluate the toxicity profile of 2-Ethylhexylamine after inhalation exposure, groups of ten male and ten female Wistar rats per test group were exposed nose-only to dynamic atmosphere of 2-Ethylhexylamine for 6 hours per day on 5 consecutive days per week for 3 months (90-day study). To enable vapor atmosphere generation. The target concentrations were 5, 25 and 125 mg/m³. A concurrent control group was exposed to conditioned air. For adaptation to the experimental conditions all animals were kept in glass restraining tubes identical to those used in the main study and were exposed nose-only to fresh air on three days before start of the exposure period.

Clinical observation was performed at least three times on exposure days and once a day during pre-exposure and post exposure observation days. Body weight and food consumptions were determined for all animals. Detailed clinical observation was performed three times (prior to start, at midterm and against the end of the exposure). Ophthalmological examinations were performed from the animals prior to exposure and towards the end of the exposure. Functional observation battery and motor activity test were performed on study day 77. Oestrus cycle was determined in all female animals from study day 69 onward.

After the last exposure, blood was sampled, hematology and clinical chemistry parameters were determined. The animals were then subjected to gross necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). Histological examinations were performed according to respective test guidelines. In addition, sperm parameters in testis and epididymidis were assessed.

RESULTS

The following substance-related adverse effects were observed in the lungs:

Test group 3 (125 mg/m³)

·        Lower mean body weight of the male animals on several days during exposure from day 51 onward

·        Lower body weight change of the male animals

·        Lower food consumption of the male animals

·        Minimal to moderate degeneration/ regeneration of the transitional epithelium of level I of the nasal cavity in 9/10 male and 9/10 female animals

·        Minimal degeneration/ regeneration of the transitional epithelium of level II of the nasal cavity in 5/10 male animals

 Test group 2 (25 mg/m³) and test group 1 (5 mg/m³)

·        No treatment-related adverse findings

 

CONCLUSION

Inhalation exposure of 5, 25 and 125 mg/m³ 2-Ethylhexylamine for 90 days (65 exposures) caused clearly treatment-related adverse histological changes in the nasal cavity levels I and II. Moreover, slight but significantly lower body weight and body weight gain was observed in male animals of the high concentration group (125 mg/m³). No systemic effect were observed. A No Observed Adverse Effect Concentration (NOAEC) was 25 mg/m³ under the current study condition.