(E)-4-Ethoxy-1,1,1-trifluoro-3-buten-2-one (stabilized with BHT)

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 Nov. 2006-03 Jan. 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was conducted according to an internationally recognised method, and under GLP. The substance is adequately characterised with its purity. Therefore full validation applies.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

12 May 1981

Deviations:

no

Remarks:

The report mentions no deviation from the version of the guideline in place at that time, but some deviations from the study plan (see in the field "Any other information on materials and methods incl. tables").

GLP compliance:

yes (incl. QA statement)

Remarks:

2006-12-19

Limit test:

no

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

N.B. This batch BWG 60000 was used in several studies with some descriptive information diverging slightly between the reports (for further details, see below in this field and in the following field).

- Appearance:
* Clear yellowish liquid was mentioned in the 2007 and 2008 Notox reports dealing with physico-chemical properties and in the in 2007 Solvay Pharma report dealing with ASRI test.
* Yellow liquid was mentioned in the 2006 and 2007 TNO reports dealing with toxicological endpoints.
- Storage conditions:
* Storage in freezer (=< -15°C) in the dark under nitrogen was mentioned in the 2007 and 2008 Notox reports dealing with physico-chemical properties.
* Storage in freezer (< -10°C) was mentioned in the 2007 Solvay Pharma report dealing with ASRI test.
* Storage at < -18°C (with or without the mention "protected from light") was mentioned in the 2006 and 2007 TNO reports dealing with toxicological endpoints.
- Stability under storage conditions: Stable.

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as test system, because this species is routinely used at the testing facility for this type of studies and accepted by the relevant authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animal used were outbred Wistar rats obtained from a colony maintained under SPF-conditions at Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: Not specified in the report.
- Age at study initiation: 7 weeks old.
- Weight at study initiation: 198 g and 158 g for males and females, respectively (the initial body weight variation did not exceed +/- 20 % of the mean weight for each sex).
- Fasting period before study: No information on fasting period before study, but information during exposure: no access to feed or water.
- Housing: The animals were housed under conventional conditions in macrolon cages with bedding of wood shavings (Espen E-001, ABEDD, Köflach, Austria).
- Diet: Food was provided ad libitum except during exposure.
- Water: Drinking water was provided ad libitum except during exposure.
- Acclimation period: Yes; upon arrival, the rats were taken in their unopened shipping containers to quarantine room and checked for overt signs of ill health and anomalies. Serological investigation of their microbiological status was conducted in randomly chosen rats of the lot delivered. On 17/11/2006, after the results of serology were satisfactory, the rats were moved to another animal room and were further acclimatized to the laboratory conditions. The duration of the acclimatization period until the experimental start date was 5 days.

DETAILS OF FOOD AND WATER QUALITY:
- Food: All rats were fed a commercially available rodent diet (Rat & Mouse No. 3 Breeding Diet RM3) from SDS Special Diets Services, Witham, England. Each batch of this diet was analysed by SDS for nutrients and contaminants. The certificate of analysis pertaining to the batches used were appended to the report. During the study, the feed was provided as a powder, in stainless steel cans.
- Water: Each cage was supplied with domestic mains tap-water suitable for human consumption (quality guidelines according to Dutch legislation based on EC Council Directive 98/83/EC). The water was given in polypropylene bottles, which were cleaned weekly and filled as needed. Results of the routine physical, chemical and microbiological examination of drinking water as conducted by the supplier were made available to the test facility. In addition, the supplier periodically (twice per year) analysed water samples taken at the premises of TNO in Zeist for a limited number of physical, chemical and microbiological variables.The results of the samples taken during or close to the conduct of this study were appended to the report.

ENVIRONMENTAL CONDITIONS
- Temperature: Between 20 and 24°C.
- Humidity: Generally between 40 and 70%, but above this range on several days for short periods associated with cleaning activities (highest value noted: 81%).
- Air changes: ca. 10 per hour.
- Photoperiod: 12 hours light / 12 hours dark cycle.

IN-LIFE DATES:
- Start of study: 22/11/2016.
- Termination of study: 20/12/2006 (necropsy of animals from the main study), 03/01/2007 (necropsy of animals from the recovery study).

Administration / exposure

Route of administration:

inhalation: vapour

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks:

The vapour was transported to the inhalation chamber by a mass flow controlled flow of humidified air.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed to the test atmosphere in a nose-only inhalation chamber, a modification of the chamber manufactured by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom. The inhalation chamber consisted of a cylindrical PVC column, surrounded by a transparent hood. The column had a volume of ca. 70 L with 45 ports for animal exposure.
- Method of holding animals in test chamber: The animals were secured in plastic animal holders (Battelle), positioned radially through the outer hood around the central column.
- Source and rate of air / Method of conditioning air: The inhalation equipment was designed to expose rats to a continuous supply of fresh test atmosphere. For each exposure chamber, test material was passed using a motor­driven syringe pump (WPI Type SP22i, World Precision Instruments, Sarasota FL, USA) to a glass evaporator. The glass evaporators were held at a constant temperature with a thermostated water bath. The vapour was transported to the inhalation chamber by a mass flow controlled flow of humidified air. The resulting test atmosphere entered the exposure chamber at the bottom and was exhausted at the top. The settings of the pumps and the reading of each mass flow controller were recorded prior to the start of each exposure. Moreover, the readings of the mass flow controllers were checked approximately every two hours.
- Temperature in air chamber: The average temperature during exposure was 22.7, 22.2, 21.8 and 22.3°C for the control and the low, mid and high concentration test atmospheres, respectively. Temperature was within the 20-24°C range, except for the first 6 minutes of the exposure of one group on 5 December 2006, the lowest temperature measured was 19.8°C.
- Humidity in air chamber: The average relative humidity during exposure was 43, 37, 49 and 46% for the control and low, mid and high concentration test atmospheres, respectively. Relative humidity was within the 30-70% range.
- Pressure in air chamber: Not reported.
- Air flow: The mean airflow through the exposure chambers during exposure was 27.1, 43.4, 15.9 and 27.0 L/min for the control, low, mid and high concentration test atmospheres, respectively.
- Method of particle size determination: Because the test material was generated as a vapour, particle size measurements were not carried out.
- Treatment of exhaust air: Not reported.

TEST ATMOSPHERE
- Brief description of analytical method used: The concentration of ETFBO in the test atmospheres was measured by infrared photoacoustic absorption analysis (Dual gas monitor, type 3426, Brüel & Kjaer, Denmark). With a period of about 80 s, the analyser sampled test atmosphere and analysed the absorption. With a cycle of 15 min, the samples were drawn consecutively from the four exposure units. The mean response for each exposure was calculated by averaging values read every 5 min. The output of the analyser was calibrated. After calibration, the calibration equations were used to convert the readings of the analyser to test atmopshere concentrations of ETFBO.
- Samples taken from breathing zone: Test atmopshere samples were taken at the animals' breathing zone in the exposure unit.

Analytical verification of doses or concentrations:

yes

Remarks:

See above in the field "Details on inhalation exposure / TEST ATMOSPHERE / Brief description of analytical method used".

Duration of treatment / exposure:

Exposure duration: 6 hours/day.

Frequency of treatment:

Exposure frequency: 5 days/week during a period of 4 weeks resulting in a total number of 20 exposure days.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.1 ppm

Remarks:

Low concentration test atmosphere
Mean actual concentration (+/- standard deviation) = 0.08 (+/- 0.08) ppm

Doses / concentrations 2

Dose / conc.:

0.3 ppm

Remarks:

Mid concentration test atmosphere
Mean actual concentration (+/- standard deviation) = 0.27 (+/- 0.09) ppm

Doses / concentrations 3

Dose / conc.:

1 ppm

Remarks:

High concentration test atmosphere
Mean actual concentration (+/- standard deviation) = 0.91 (+/- 0.07) ppm

No. of animals per sex per dose:

The 4-week study comprised four main groups of 5 males and 5 females (one control group and three test groups which were exposed to the three different concentrations of the test material). Two recovery groups, also consisting of 5 males and 5 females each, were also used (one control group and one test group exposed to the high concentration test atmosphere).

Control animals:

yes

Details on study design:

- Dose selection rationale: The concentration levels were established based on a preceding acute inhalation toxicity study (see IUCLID section 7.2.2: TNO study 7221/01, 2007) in which the 4-h LC50 value was estimated to be between 8.2 and 12.3 ppm for both sexes.
- Animal assignment: One day before the start of exposure, 30 males and 30 females were allocated to the various groups by computer randomization, taking their body weights into account.
- Fasting period before blood sampling for clinical biochemistry: Yes, rats were fasted overnight before blood sampling at necropsy.
- Rationale for selecting satellite groups / Post-exposure recovery period in satellite groups: Two additional groups (5 males and 5 females each) were exposed to the control and high concentration test atmosphere to investigate recovery during a 14-day post-exposure period.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. A group-wise observation was made halfway through each exposure day. On working days, all cages were checked again in the afternoon. On week-end days and public holidays, only one check per day was carried out.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were examined during cage side observations (see above).

BODY WEIGHT: Yes
- Time schedule: The body weight of each animal was recorded once during the acclimatization period in order to monitor adequate growth (day -5), one day before the start of exposure (day -1), at initiation of treatment (day 0) and weekly thereafter. In addition, animals were weighed before sacrifice for calculation of relative organ weights.

FOOD CONSUMPTION & EFFICIENCY: Yes
- Time schedule: Food consumption was measured per cage, over three successive 7-day and one 6-day period for animals of the main groups. In the recovery groups, food consumption was measured similarly, over five successive 7-day and one 6-day period. The results were expressed in g per animal per day. The efficiency of food utilization was calculated and expressed in g weight gain per g food consumed.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
* Main study: At the end of the exposure period, in blood samples taken at necropsy (nominal day 28) from the abdominal aorta.
* Recovery study: At the end of the 14-day recovery period.
- Anaesthetic used for blood collection: Yes (Nembutal® anaesthesia). In addition, K2-EDTA was used as anticoagulant.
- Animals fasted: Yes (overnight).
- How many animals: All animals of the main study and recovery groups.
- Parameters examined:
* haemoglobin
* packed cell volume
* red blood cell count
* reticulocytes (not examined during the recovery study)
* total white blood cell count
* differential white blood cell count
* prothrombin time (not examined during the recovery study)
* thrombocyte count
- Parameters calculated:
* mean corpuscular volume (MCV)
* mean corpuscular haemoglobin (MCH)
* mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
* Main study: At the end of the exposure period at the same time blood samples for haematology were collected.
* Recovery study: At the end of the 14-day recovery period.
- Animals fasted: Yes (overnight).
- How many animals: All animals of the main study groups, only female of the recovery study groups.
- Parameters examined:
* alkaline phosphatase activity (ALP) (not examined during the recovery study)
* aspartate aminotransferase activity (ASAT) (not examined during the recovery study)
* alanine aminotransferase activity (ALAT) (not examined during the recovery study)
* gamma glutamyl transferase activity (GGT) (not examined during the recovery study)
* total protein
* albumin
* ratio albumin to globulin
* urea (not examined during the recovery study)
* creatinine (not examined during the recovery study)
* fasting glucose (not examined during the recovery study)
* bilirubin total (not examined during the recovery study)
* cholesterol (not examined during the recovery study)
* triglycerides (not examined during the recovery study)
* phospholipids (not examined during the recovery study)
* calcium (not examined during the recovery study)
* sodium (not examined during the recovery study)
* potassium
* chloride (not examined during the recovery study)
* inorganic phosphate (not examined during the recovery study)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, at the end of the exposure (main study) / recovery (recovery study) period, all animals were killed. The following organs were weighed (paired organs together) as soon as possible after dissection (to avoid drying):
* adrenals
* heart
* kidneys
* liver
* spleen
* testes
* lungs with trachea and larynx

HISTOPATHOLOGY: Yes, samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde (10% solution of formalin). The lungs (after weighing) were infused with the fixative under ca. 15 cm water pressure to insure fixation.
* adrenals
* heart
* kidneys
* liver
* spleen
* testes
* complete respiratory tract including nasal passages
* all relevant gross lesions
- Preparation of slides: The respiratory tract of all animals, and all other organs/tissues of animals of the control and high concentration main study groups were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
- Histopathological examination: The organs of the list above were examined in animals of the main high concentration group and main control group. The nasal tissues were examined at 6 levels, the larynx at 3 levels, the trachea at 3 levels (including the bifurcation), and each lung lobe at 1 level. Because histopathological changes were found in the respiratory tract of animals of the high concentration group at the end of the exposure period, the respiratory tract of animals of the intermediate groups and the recovery groups was also examined.

Statistics:

The statistical procedures for analysis of data are described below and in the field "Any other information on materials and methods incl. tables":
- Pre-treatment body weight data, clinical pathology data and organ weights: one-way analysis of variance (Anova) after checking for homogeneity of variances (Bartlett test) and normality of data distribution (Shapiro-Wilks test). If variances were not homogeneous or data were not normally distributed, the data were stepwise log or rank transformed prior to the Anova. If the Anova showed a significant effect (p<0.05), intergroup comparisons with the control group were made by Dunnett's multiple comparison test.
- Body weight data collected after initiation of treatment: one-way analysis of covariance (covariate:pre-treatrnent body weight values) using an automatic Decision Tree. The Decision Tree process is summarized as follows:
(1) Data were tested for homogeneity of variance (Bartlett test) and normality of data distribution (Shapiro-Wilks test). A 'best ' transformation of the data was determined (none, log or rank), defining whether the results should be analysed parametrically or non-parametrically. The use of covariates was checked (if this led to removal of the covariate, non-parametric pairwise comparisons were conducted), and the homogeneity of group means was assessed by an F-ratio test.
(2) The (transformed) data were analysed to find the lowest concentration level that showed an effect using the Williams test for parametric data or the Shirley test for non-parametric data.
(3) If no trend effect was found, but the data showed non-homogeneity of group means in step (1) then the data were analysed by a Dunnett (parametric) or Steel (non parametric) test to look for significant differences between the control and treatment groups.
- Food consumption: Dunnett's multiple comparison test.
- Incidences of histopathological changes: Fisher's exact probability test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males: No observations reported.

Females: Although observation of the rats was limited during exposure due to the stay in restraining tubes, it was noted about halfway each exposure day that females rats of the high concentration group were restless and wet, or wet only.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Males: No mortality observed.

Females: One female of the mid concentration group was found dead on 18 December 2006, a few hours after exposure had ended on that day. This animal was found dead accidentally getting trapped between the feeder and the cage rack.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males: Body weight gain was clearly less in males of the high concentration group, reaching statistical significant levels on days 7, 14 and 27 of the study. Body weight gain recovered slightly during the 14-day post-exposure period.

Females: Female animals of the high concentration group showed a slight, but statistically significant lower body weight on day 21 only.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males: Food intake was low in males of the high concentration group from day 7 to 27 when compared to the control group. Food intake during the 14-day post-exposure period was comparable to that of controls; both groups showed a higher food intake than during the exposure period.

Females: Food intake during the exposure period was comparable between the groups. Female animals also showed a higher food intake during the post­exposure period when compared to the exposure period.

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Males:
- The number of thrombocytes was lower in animals of the high concentration group compared to controls, reaching a statistically significant Ievel in male animals only.
- The total number of white blood cells was significantly decreased in males of the high concentration group, which was reflected in a significantly decreased absolute number of lymphocytes, and in a decreased relative number of lymphocytes and an increased relative number of neutrophils.
- Male animals of the low, mid and high concentration groups showed a slight, though statistically significant decrease in the absolute number of eosinophils, which was not reflected in changes in the relative number. Because these changes were limited, and these numbers in females were increased rather than decreased, these were considered to be incidental findings.
- No changes were seen in the recovery groups.

Females:
- The number of thrombocytes was lower in animals of the high concentration group compared to controls; however a statistically significant Ievel was not reached in females.
- At the end of the 14-day post-exposure period, female animals of the high concentration group showed an increased MCV and a decreased MCHC value but no other changes in red blood cell parameters. The increase in MCV and the decrease in MCHC were not considered biologically relevant due to the absence in changes in the underlying parameters such as red blood cell numbers, PCV and haemoglobin content.
- A decreased relative number of lymphocytes and an increased relative number of neutrophils were also observed in female rats of the high concentration group which had resulted in a significantly increased number of neutrophils only.
- No changes were seen in the recovery groups.

Summary and discussion:
Changes in red and white blood cell parameters were observed consisting of decreases in the number of thrombocytes and increases in the number of neutrophils (both sexes). The increases in neutrophil count in blood as seen in males and females of the high concentration group were not similarly reflected in related parameters in rats of both sexes. In males, the increase in the relative number of neutrophils and the decreases in the relative number of lymphocytes resulted in decreases in the absolute number of lymphocytes, with a concomitant decrease in the absolute white blood cell number. In females, in contrast, the increase in the relative number of neutrophils and the decreases in the relative number of lymphocytes resulted in increases in the absolute number of neutrophils only. The changes mentioned above point at local damage to the respiratory tract and histopathological examination did confirm this. The changes in blood such as the increases in neutrophils may therefore be secondary to the initial exposure.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
No effects reported.

Females:
- A statistically significant decrease in the albumin/globulin ratio (but not in the albumin and globulin concentrations) was observed in females of the high concentration group at the end of the exposure period and at the end of the 14-day post-exposure period. These decreases were not considered of biological relevance due to the absence in changes in the underlying parameters albumin and globulin.
- Females of the low, mid and high concentration groups, in addition, showed a decreased potassium content, which was not clearly concentration-related and therefore considered an incidental finding.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males:
- Absolute and relative lung weights were increased in males of the high concentration group. Statistically significant levels were reached in relative weight.
- Absolute but not relative kidney and liver weights were decreased in males of this group. The decreases in the absolute liver and kidney weights were explained by the decreased body weights of these animals.

Females:
- Absolute and relative lung weights were increased in females of the high concentration group. Statistically significant levels were reached in relative and absolute weight.
- An increased absolute heart weight was observed in female rats of the high concentration group at the end of the 14-day post-exposure period. Because the relative heart weight had not changed, this finding was not considered to be of toxicological relevance.

Summary:
Exposure-related organ weight changes were observed for lungs and consisted of increases in absolute and relative lung weight in animals of both sexes.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Males & females - Main study: At necropsy, two males and two females of the high-concentration group had poorly collapsed lungs. This was most probably exposure-related.

Males & females - Recovery study: At necropsy, no treatment related macroscopic changes were observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Males & females - Main study:
- Microscopic examination revealed treatment related histopathological changes in the nose, the larynx, the trachea and the lungs of the high-concentration animals. Especially the respiratory epithelium in the entire respiratory tract appeared to be target for the test material. The alveoli in the lungs and the olfactory epithelium in the nasal cavity appeared to be quite resistant to damage. These findings were consistent with the acute study (see IUCLID section 7.2.2: TNO study 7221/01, 2007).
- The histopathological changes in the nasal cavity were summarized as slight to moderate rhinitis and were characterised by the following: the respiratory epithelium showed loss of ciliae and hyperplasia. The top layers of the hyperplastic cells often were flattened (squamous metaplasia). The number of goblet cells was decreased. In the lamina propria infiltration of mononuclear inflammatory cells was observed. Exsudate containing polymorphonuclear inflammatory cells was present in the nasal lumen. The histopathological changes in the nasal cavity were predominantly present in the rostral parts, mainly in nasal level 2 and to a somewhat lesser extent in nasal level 3. Apart from the presence of some luminal exsudate the caudal parts of the nasal cavity showed hardly any histopathological changes. An exception was observed in one high-concentration animal, which also showed inflammatory infiltration and focal loss of olfactory epithelium in nasal level 5.
- The larynx and trachea of the high concentration animals showed treatment related histopathological changes comprising squamous metaplasia (mainly in the larynx) and/or epithelial hyperplasia (mainly in the trachea), often accompanied by mononuclear cell infiltrate. The changes in the trachea were generally more prominent in the upper part than in the lower part. The larynx and trachea were slightly more affected in the females than in the males. Minimal focal mononuclear cell infiltrate in the rat larynx and trachea can also occur as background findings, but in the present study the incidence was clearly increased due to exposure to the test material.
- The respiratory epithelium of the airways in the lungs of the high-concentration animals showed a variety of histopathological changes, summarised as respiratory epithelial changes. These consisted of areas witb loss of ciliae, epithelial hypertrophy, decreased number of non-ciliated bronchiolar epithelial cells (Clara cells), and/or areas with a flattened epithelial layer, which was often present at the site of the bronchus associated lymphoid tissue (BALT). The severity was minimal, however, large areas of the airways were involved. The various histopathological changes occurred in a variety of combinations but it is likely that they all represent different stages of the response to the exposure of the lungs to the test material. Further, in some high-concentration animals the presence of (BALT) was more prominent than in the lungs of the controls. The alveoli were not affected in any of the animals. The morphological changes of the respiratory epithelium of the airways may explain the incomplete collapse of the lungs observed at necropsy in some high-concentration animals.
- All mid-concentration males and three mid-concentration females demonstrated squamous metaplasia in the larynx. Although the incidence in the females did not reach the level of statistical significance, this finding was considered related to treatment, as in the males.
- The histopathological changes observed in all other organs were considered incidental findings, not related to treatment.

Males & females - Recovery study:
- The nasal cavity in the high-concentration animals showed signs of recovery, however, all animals still showed a very slight chronic inflammatory reaction mainly in the maxilloturbinates of nasal level 2. The inflammatory reaction was characterised by fibrosis, mononuclear cell infiltrate and sometimes a thickened epithelial cell layer. The severity of each of these changes was minimal but together they caused slight thickening of the maxilloturbinates.
- The larynx and trachea did not show clear exposure-related hispathological changes anymore, although the squamous metaplasia in two high concentration males was suggestive of a remnant of the changes observed at the end of the exposure period.
- The repiratory epithelium in the lungs had recovered for the most part, but two high concentration males and four high concentration females still showed focal respiratory epithelial flattening at the site of BALT.

Summary:
- In animals of the high concentration group, exposure-related histopathological changes were observed in the complete respiratory tract at the end of the exposure period. Especially the respiratory epithelium throughout the respiratory tract appeared to be a target. Changes consisted of loss of ciliae, epithelial hyperplasia or hypertrophy, squamous metaplasia, decreased number of goblet cells, infiltration of mononuclear inflammatory cells, luminal exsudate with polymorphonuclear inflammatory cells, and a decreased number of non-ciliated bronchiolar epithelial cells (Clara cells). Respiratory tract changes were still present in animals of the high concentration sacrificed at the end of the 14-day post-exposure period, but to a lesser extent, which pointed at recovery of the effects. Apparently a 14-day post-exposure period was not sufficient for a complete recovery
- In animals of the mid concentration group, changes consisted of laryngeal squamous metaplasia only.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

Most of the exposure-related effects (clinical signs, body weight and food consumption changes, haematological findings, lung weight changes and gross pathological findings) were observed at the high concentration only. Exposure-related histopathological changes in the respiratory tract were the most important at the high concentration and were still observed at the mid concentration, but were considerably less severe. This parameter was selected for determining the NOAEL.

NOAEL conversion from ppm to mg/L:
Concentration (mg/m3) = Concentration (ppm) x Molecular weight (g/mol) / 24.5 (L/mol)
Where:
24.5 L/mol = gas constant at 25 °C and 1013.25 hPa
Concentration (ppm) = 0.1 ppm for the NOAEL of ETFBO
Molecular weight (g/mol) = 168.11 for ETFBO
Concentration (mg/m3) = 0.1 x 168.11 / 24.5
Concentration (mg/m3) = 0.69
Concentration (mg/L) = 0.00069

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

0.1 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Effect level:

0.001 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: for conversion in mg/L, see in the field "Details on results".

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.3 ppm

System:

other: respiratory system: entire respiratory tract

Organ:

larynx

lungs

nasal cavity

trachea

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Overall average actual concentrations (in ppm) of ETFBO in test atmospheres:

	Low concentration	Mid concentration	High concentration
Average (± standard deviation)	0.083 (± 0.08)	0.267 (± 0.088)	0.908 (± 0.071)

For such low levels, the values are rather near to the target concentrations of 0.1, 0.3 and 1 ppm. The high standard deviation for the low concentration is largely due to the first two exposure days with high values.

Mean body weight (± standard deviation, in g):

Groups	Day -5	Day -1	Day 0	Day 7	Day 14	Day 21	Day 27	Day 28	Day 35	Day 41
Control Males   Females	154.35 (±9.74) 135.97 (±6.91)	188.56 (±9.95) 154.43 (±5.75)	196.80 (±9.68) 157.88 (±6.30)	220.24 (±8.66) 164.93 (±7.68)	249.45 (±11.51) 171.04 (±6.54)	271.24 (±13.65) 179.31 (±5.78)	288.11 (±17.12) 185.35 (±8.42)	282.78 (±16.75) 183.62 (±11.11)	308.76 (±18.89) 198.82 (±9.56)	322.94 (±18.99) 204.44 (±8.32)
Low conc. Males   Females	152.68 (±8.80) 135.76 (±6.79)	187.18 (±10.33) 155.60 (±6.44)	195.16 (±8.10) 159.92 (±7.64)	222.86 (±9.36) 167.08 (±8.03)	249.76 (±7.82) 177.04 (±8.67)	274.10 (±6.93) 182.44 (±9.05)	290.78 (±7.04) 187.18 (±10.22)
Mid conc. Males   Females	157.68 (±8.77) 131.64 (±4.83)	195.18 (±9.18) 152.88 (±6.47)	204.60 (±6.80) 156.48 (±6.04)	224.20 (±4.62) 161.26 (±7.30)	256.40 (±9.01) 172.86 (±6.56)	278.86 (±12.64) 175.66 (±7.08)	297.68 (±13.20) 182.30 (±8.41)
High conc. Males   Females	156.01 (±8.44) 136.00 (±5.59)	189.09 (±10.17) 155.94 (±6.42)	196.56 (±10.57) 158.86 (±7.08)	212.89* (±16.94) 163.01 (±6.43)	232.73** (±21.28) 167.55 (±5.58)	249.06 (±26.79) 172.54* (±8.41)	253.47** (±29.35) 181.38 (±6.91)	253.02 (±31.96) 178.38 (±7.33)	283.60 (±37.02) 198.08 (±10.98)	300.48 (±34.89) 206.20 (±12.98)

Statistical analysis (default decision tree; covariate body weight day -1): * p < 0.05; ** p < 0.01.

Food intake (in g/rat/day):

Groups	Days 0 to 7	Days 7 to 14	Days 14 to 21	Days 21 to 27	Days 28 to 35	Days 35 to 41
Control Males Females	17.60 13.45	19.30 13.45	19.90 13.75	20.10 13.70	20.60 14.60	21.20 14.50
Low conc. Males Females	16.70 14.80	19.40 16.90	20.70 16.40	20.90 16.60
Mid conc. Males Females	16.70 13.80	19.90 14.10	20.80 13.90	21.00 13.50
High conc. Males Females	16.85 13.20	17.10 12.85	18.00 13.45	16.95 14.10	20.20 15.20	21.00 15.50

Food intake was measured per cage (5 animals/ cage).

Mean absolute and relative lung weight (± standard deviation, in g):

Groups	Absolute weight		Relative weight
	End of treatment	End of recovery	End of treatment	End of recovery
Control Males   Females	1.164 (±0.072) 0.938 (±0.045)	1.308 (±0.101) 0.944 (±0.039)	4.340 (±0.214) 5.608 (±0.201)	4.254 (±0.217) 4.866 (±0.074)
Low conc. Males   Females	1.238 (±0.052) 0.938 (±0.062)		4.646 (±0.230) 5.580 (±0.561)
Mid conc. Males   Females	1.248 (±0.111) 0.920 (±0.079)		4.610 (±0.342) 5.588 (±0.378)
High conc. Males   Females	1.268 (±0.076) 1.076* (±0.102)	1.222 (±0.112) 0.976 (±0.052)	5.250** (±0.487) 6.698** (±0.708)	4.290 (±0.175) 4.888 (±0.311)

Statistical analysis (default decision tree): * p < 0.05; ** p < 0.01

Applicant's summary and conclusion

Conclusions:

The NOAEL value of ETFBO based on histopathological changes in the entire respiratory tract was determined to be 0.00069 mg/L in male and female Wistar rats.

Executive summary:

The subacute inhalation toxicity to male and female Wistar rats was investigated in a GPL-compliant 28-day study performed according to OECD test guideline 412.

 

Groups of five male and five female rats were exposed nose-only to target concentrations of 0 ppm (control), 0.1 ppm (low concentration test atmosphere, actual concentration of 0.08 ppm), 0.3 ppm (mid concentration test atmosphere, actual concentration of 0.27 ppm) or 1 ppm (high concentration test atmosphere, actual concentration of 0.91 ppm) for six hours a day, five days a week during a four-week period, with a total number of 20 exposure days. Additional groups of five male and five female rats were exposed in a similar way to 0 ppm (control) or 1 ppm (high concentration test atmosphere) and subsequently kept for a 14-day post-exposure period. During exposure, ETFBO was generated as a vapour. To examine the toxicity of the test material, data on clinical signs, body weight gain, food consumption, haematology and clinical chemistry were recorded. In addition, animals were grossly examined at necropsy, organs were weighed, and a selection of organs and tissues (including the complete respiratory tract and nasal passages) was examined microscopically.

 

Overall, exposure of rats to 1.0 ppm ETFBO for 6 hours/day, 5 days/week for 4 weeks resulted in local effects and changes considered to be secondary to the local effects. These consisted of decreases in body weight gain (especially in males), decreases in food intake (in males), changes in blood cell parameters and increases in lung weight (in males and females). Histopathological changes were seen in the entire respiratory tract, from the nasal cavity to the lungs. In animals exposed to 0.3 ppm ETFBO, changes consisted of laryngeal squamous metaplasia only.

Most of the exposure-related effects (clinical signs, body weight and food consumption changes, haematological findings, lung weight changes and gross pathological findings) were observed at the high concentration only. Exposure-related histopathological changes in the respiratory tract were the most important at the high concentration and were still observed at the mid concentration, but were considerably less severe. Based on this pool of evidence, the NOAEL was thus set at the low concentration.

 

Under the conditions of this study, the NOAEL value of ETFBO based on histopathological changes in the entire respiratory tract was determined to be 0.1 ppm, corresponding to 0.00069 mg/L. According to the GHS criteria, ETFBO has to be considered as causing damage to respiratory organs through prolonged or repeated exposure by inhalation route and has to be classified in category 1 for STOT-RE.