Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
yes
Remarks:
Necropsy, blood collection, urine collection and body weight was performed on day 30 for G1, G2 and G3 instead of day 29.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
EC Number:
601-478-9
Cas Number:
117428-22-5
Molecular formula:
C18H16F3NO4
IUPAC Name:
methyl (2E)-3-methoxy-2-[2-({[6-(trifluoromethyl)pyridin-2-yl]oxy}methyl)phenyl]acrylate
Test material form:
solid
Details on test material:
- Purity: 93.3-99.8% (see individual test record for specific details)
Specific details on test material used for the study:
99.3% purity

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 7-9 weeks
- Weight at study initiation: mean of 184.6-204 g (males); mean of 146.8-159.0 g (females)
- Housing: Rats were housed in standard polypropylene cages group wise and sex wise, each cage containing three rats for range finding study and five rats for main study. Enrichment materials (Tunnels and Crawl balls) were used.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): reverse osmosis ad libitum
- Acclimation period: 7 days; rats were acclimatized to the restraint tubes for a short period during the acclimation period prior to testing.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.2 - 23.8°C
- Humidity (%): 43.2 – 61.5%
- Air changes (per hr): Not reported
- Photoperiod (hrs dark / hrs light): 12 hour light and 12 hour dark

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
> 1 - < 3 µm
Geometric standard deviation (GSD):
1
Remarks on MMAD:
Aerosol size measurements were collected in the pilot study to confirm respirability.
Details on inhalation exposure:
Prior to the main study, a range finding study where 3 male and 3 female rats were exposed nose only to 0.50 mg/L of the test substance was performed. The 0.50 mg/L target concentration was based upon a previously reported LC50 values of >2.12 mg/L for male and 3.19 mg/L for female rats. Two females exposed to 0.50 mg/L died within 2 hours of exposure while all other animals were euthanized without necropsy on day 3 post-exposure. A second range finding study was performed to select the tolerable concentration levels for the repeated exposure study. Three male and three female rats were exposed to 0.025 mg/L of the test substance, 6 hours/day, for five days with two additional days of observation. All the rats tolerated the 0.025 mg/L exposure level for five days. Therefore, the target concentration levels of 0.001, 0.01 and 0.025 mg/L were selected for the low, intermediate and high concentration groups, respectively for the repeated exposure main study.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
6 hours a day, 5 days a week
Frequency of treatment:
4 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0.001 mg/L air (nominal)
Dose / conc.:
0.01 mg/L air (nominal)
Dose / conc.:
0.025 mg/L air (nominal)
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor.
Positive control:
No

Examinations

Observations and examinations performed and frequency:
Six groups were included in this study: Group G1 was the control group; Group G2 was the low-dose group (0.001 mg/L); Group G3 was the intermediate-dose group (0.01 mg/L); Group G4 was the high-dose group (0.025 mg/L); Group G5 was the Sattelite group control; and Group G6 was the Exposed Satellite group (0.01 mg/L).

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All the rats were observed individually during and after the exposure and daily throughout the experimental period. Cage-side observations included changes in the skin and fur, eyes, and mucous membranes; changes in respiratory and circulatory systems, changes in nervous system, and changes in somatomotor activity and behavior patterns. Attention was directed to observation of tremors, convulsions, salivation, diahorrea, lethargy (dullness), sleep and coma.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During and after exposure

BODY WEIGHT: Yes
- Time schedule for examinations: Individual animal body weights were measured on day 0, 3, 7, 10, 14, 17, 21, 24, 28, 29 and 30. The satellite group (G5 and G6) of animals were weighed additionally on day 31, 35, 38, 42 and 43.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes; cage wise feed consumption was recorded daily and reported weekly.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: cage wise water consumption was recorded daily and reported weekly.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: All
- Parameters examined: RBC count, WBC count, hematocrit (HCT, PCV), platelet count, hemoglobin (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), differential count (5 parts) using a Hematology Bayer Advia 120 fully automated analyzer. Prothrombin time was measured by using CoaData 2001 coagulometer. Reticulocyte count and clotting time were done by manual method.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: G1 - G4 : Day 0 & 29 and/or Day 30; G5 - G6 : Day 0, 29 and Day 43
- Animals fasted: Yes
- How many animals: All
- Parameters examined: Glucose, total cholesterol, triglycerides, total bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP, SAP), blood urea nitrogen (BUN), urea, creatinine, total protein, albumin, globulin, phosphorus and calcium levels were analysed using Siemens Dimension Xpand fully automated biochemistry analyzer. Sodium, chloride and potassium were analyzed by Human Humalyte electrolyte analyzer.

URINALYSIS: Yes
- Time schedule for collection of urine: Urine was collected and pooled group and sex wise in labeled, clean, sterile, wide mouth containers sealed with tightly fitting lids on days 0 and 29 or 30 of all rats belonging to Groups G1- G6. Urine from animals belonging to G5 and G6 were collected on day 43.
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: 1. Color – Visual observation 2. pH – Multistrip 3.Specific gravity – Multistrip 4. Sediment – Visual observation 5. Albumin – Multistrip 6.Glucose – Multistrip 7. Bilirubin – Multistrip 8.Ketones – Acetone – Multistrip 9.Blood – Multistrip 10.WBC – Microscopic 11. RBC– Microscopic 12.Epithelial cells– Microscopic 13.Casts– Microscopic 14.Crystals– Microscopic 15.Organisms– Microscopic 16.Abnormal constituents – Microscopic.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

The following organs were collected and weighed wet as soon as possible after dissection: Brain, Heart, Lungs, Liver, Kidneys, Epididymides, Spleen, Thymus, Adrenals, Testes, Ovaries, and Uterus.

The following organs/tissues were collected from all animals and preserved in 10% neutral buffered formalin. Testes and eyes will be pre-fixed with Modified Davidson’s and Davidson’s fixative, respectively for 48 hours, washed in running tap water for 5 minutes and were transferred into 10% neutral buffered formalin. The lungs were removed intact, weighed as mentioned above and inflated with 10% neutral buffered formalin: Heart & Aorta, Spleen, Prostate, Nasopharyngeal tissues, Liver, Thymus, Epididymides, Trachea, Kidneys, Brain, Mammary Gland, Lungs, Pituitary, Larynx, Lacryminal gland, Lymph nodes (Mediastinal, Maxillary, Mesenteric, and Tracheobronchial), Sciatic nerve, Eyes, Salivary gland, Adrenals, Testes, Esophagus, Stomach, Ovaries, Skin, Small Intestine, Uterus, Thyroid with parathyroid, Bone marrow (sternum), Large intestine, Pancreas, Urinary bladder, Spinal cord (cervical, mid-thoracic, and lumbar), and Seminal vesicles.

All test animals were subjected to a detailed gross pathological examination. The tissues from all the groups in the above list were subjected for histopathology examination. These organ/tissue samples were processed, embedded with paraffin wax, sectioned at approximately 5 μm thick and stained with hematoxylin and eosin. The histological sections of the respiratory tract were taken as follows: Nasopharyngeal tissues (Four levels: posterior upper incisor, incisive papilla, second palatine crest, and first molar teeth levels), Trachea (Two levels: Transverse and Longitudinal horizontal), Larynx (Three Levels: Base of epiglottis, Ventral pouch, and Cricoid cartilage), and Lungs (Sections of all five lobes).
Statistics:
The data was subjected to Modified-Levene equal-variance test for homogeneity. Homogenous data was submitted to Analysis of Variance (ANOVA) followed by Student-Newman-Keul’s test for post hoc comparison. Heterogeneous data was subjected to non-parametric tests (NCSS 2007 statistical software). Data for group 2 (0.001 mg/L), group 3 (0.01 mg/L) were compared to the group 1 air-exposed control group. Data for the group 4 (0.025 mg/L) and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group. The organ weight data for groups 2 (0.001 mg/L), group 3 (0.01 mg/L) and 4 (0.025 mg/L) was compared to group 1 air-exposed control group and group 6 (0.01 mg/L satellite) was compared to the group 5 air-exposed control satellite group.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Female rats exposed to 0.0017 mg/L (low-dose) and 0.025 mg/L (high-dose) demonstrated statistically significant increases in body weights when compared to the control on test days 10 and 28, respectively. Since these changes were increases and not decreases they were considered spurious, non-adverse and not test substance-related. Female rats exposed to 0.012 mg/L (satellite group) demonstrated statistically significant increase in body weights on test days 21, 28, 29, 31 and 35 when compared to the respective control group values and were considered to be non-adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no exposure-related differences in haematology or coagulation parameters in male or female rats following the treatment or recovery periods.

Mean corpuscular hemoglobin (MCH) was minimally decreased at test day 29 in high-dose females. A decrease in reticulocyte count (RC) was also seen at test day 29 in high-dose group females. The minimal decrease in MCH and RC occurred in the absence of clear correlation with other red cell indices. Therefore, this minimal decrease in MCH and RC was considered unrelated to treatment and non-adverse.

Clotting time was minimally decreased in high-dose group females at test day 29. Similar change in clotting time was not observed in male rats. This change was likely spurious and unrelated to treatment. In addition minimal decrease in clotting time has no toxicologic significance; therefore, this change was considered to be non-adverse.

The following statistically significant changes in mean haematology or coagulation parameters were considered unrelated to treatment because they either only occurred during pretest or were not dose related: On day 0 (Pre-test), there were an increases in the eosinophils and MCV and decreases in the clotting time and WBC counts of high-dose group females when compared to the satellite Control group; On day 30, males of low-dose group showed an increase in clotting time when compared to the control group; On day 29, satellite group females showed an increase in monocytes when compared to the satellite control group.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they only occurred during pretest: On day 0 (Pre-test), an increase in potassium levels of satellite group males when compared to the satellite control group.

The following statistically significant changes in mean clinical chemistry parameters were considered unrelated to treatment because they were not dose related or individual values in these groups were in range of the historical control data: On day 29, an increase in glucose, calcium and sodium concentration were observed in the high-dose group males when compared to the satellite control group. These parameters were well within the historical control data. Current historical range in males for glucose is 77 - 126 mg/dL, for calcium is 7.9 - 12.7 mg/dL and for sodium is 139 - 149 mmol/L; On day 29, an increase in the glucose levels were observed in the high-dose group females when compared to the satellite control group. However, the mean was within the current historical range (74 - 127 mg/dL).

The following statistically significant change in group mean biochemical parameters in the two-week recovery groups were unrelated to treatment because similar changes were not observed at the end of the 28-day exposure period: A decrease in phosphorus and potassium levels of satellite group males was observed when compared to the satellite control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in group mean urinalyses parameters in male or female rats following the treatment or recovery periods.
Urinalysis performed on day 0, days 29/30 and day 43 did not show any treatment-related changes in any of the groups. The physical parameters like color, appearance, specific gravity, sediment, pH or biochemical parameters like glucose, albumin, bilirubin, ketone bodies and the microscopic parameters like blood, leukocytes, erythrocytes, epithelial cells, casts and crystals in treated groups were comparable with control animals and were well within the historical control data. No abnormal consituents or organisms were observed in the urinalysis.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related organ weight changes were observed in the treated animals.

The following statistical differences observed in the absolute and relative organ weights of treated animals were considered incidental and unrelated to exposure of test substance because they were unaccompanied by correlated morphologic findings or did not occur in dose dependent manner or were seen only in one sex: an increase in mean absolute and mean relative (%body weight) liver weights in G3 males (0.01 mg/L); an increase in mean absolute liver weights in G3 females (0.01 mg/L); an increase in mean absolute and mean relative (%body weight) epididymides weights in G2 (0.001 mg/L) and G3 (0.01 mg/L) males; an increase in mean absolute spleen weight in G3 males (0.01 mg/L); a decrease in mean absolute heart and adrenals weight in G4 males (0.025 mg/L); and an increase in mean relative (%body weight) brain weight in G4 females (0.025 mg/L).

In recovery groups, statistical differences were observed in some absolute and relative organ weights of treated animals as compared to recovery controls. These organ weight differences in the recovery animals were interpreted to be spurious and unrelated to exposure of test substance since organ weight effects were observed only in one sex or not observed in the main group males or females. These included: a decrease in mean absolute and relative (%body weight) spleen weights in females; an increase in relative (%body weight) brain weight in males and decreased relative (%body weight) brain weight in females and an increase in mean absolute uterus weight in females.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related gross pathological findings were observed in treated animals. The gross findings observed in this study were consistent with spontaneous findings of the type routinely observed in Wistar rats of this age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No test substance related histopathological findings were observed in exposed animals.

A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. In normal mature rats, the outer nuclear layer (ONL) in the mid-posterior region of the retina generally consists of 10-11 photoreceptor nuclei. In the mild retinal degeneration observed, the ONL was decreased to 6-9 nuclei, but the retinal layer architecture appeared normal except for some pyknotic nuclei in the inner nuclear layer. With increased in severity of retinal degeneration, the width of the ONL was further decreased and the photoreceptor segments were degenerated. When the outer retinal layers were degenerated to the 0-1 layer, the inner retina was abutted directly on the retinal pigment epithelium (RPE). Other histopathology changes in the retina included pyknotic nuclei in the inner nuclear layer. With few exceptions, inner retinal layers (nerve fiber layer, ganglion cell layers, and inner plexiform layers) were relatively unaffected, even in cases of observed marked retinal degeneration. Retinal degeneration was observed mostly in the mid-posterior region, while changes in the mid-peripheral region were minimal.

The histopathology changes observed in retina were not considered to be test substance related effects based on the following observations:

● Retinal lesions were observed in all groups (except G2), including controls, and in all rats in the control recovery groups in this study.
● In previous chronic oral study with the same test material, no test substance related retinal changes were present at any dose.

In conclusion, the retinal lesions observed in this study were not considered to be indicative of target organ toxicity to the eye. All other histopathology findings observed in this study were interpreted to be spontaneous changes of the type routinely observed in Wistar rats of this age.
Histopathological findings: neoplastic:
no effects observed
Details on results:
Rats from the low target concentration (0.001 mg/L) group (G2) were exposed to 0.0017 ± 0.00022 mg/L of the test substance (Mean ± SD) which was characterized by a MMAD (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration (0.01 mg/L) group (G3) were exposed to 0.012 ± 0.0025 mg/L of the test substance with a MMAD (GSD) of 2.15 μm (2.77). Rats from the high target concentration (0.025 mg/L) group (G4) were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD (GSD) of 2.12 μm (2.79). Rats from the intermediate-satellite target concentration (0.01 mg/L) group (G6) were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD (GSD) of 2.61 μm (2.84).

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
0.025 mg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects at highest concentration tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.
Executive summary:

Groups of 5 male and 5 female rats each were exposed nose-only, 6 hours per day, 5 days/week to target concentrations of 0 (air control), 0.001, 0.01 or 0.025 mg/L of the test substance for a total of 20 exposures. To determine reversibility of potential adverse effects satellite groups of 5 male and 5 female rats each were exposed to 0 (air control) or 0.010 mg/L of the test substance for a total of 20 exposures and were allowed to recover for 2 weeks. Test atmospheres were generated by suspending the of the test substance in air with a Wright dust feeder. Atmospheric concentrations of test substance were determined by gravimetric analysis and the mass median aerodynamic diameter (MMAD) and gravimetric standard deviation (GSD) of the aerosol atmosphere was determined with a seven stage Mercer style cascade impactor. Animals were observed for clinical signs of toxicity daily, had body weights measured twice weekly and feed and water consumption determined daily and reported weekly. Three to four days following the final exposure the air-control, low, intermediate and high concentrations groups of 10 rats (5 male and 5 female) each were fasted overnight, blood and urine samples collected and sacrificed for anatomic pathology evaluation including microscopic tissue evaluation. Three days following exposure the air (control) satellite and the intermediate satellite groups of 10 rats (5 male and 5 females) each were fasted overnight, blood and urine samples collected. Two weeks following exposure the air (control) satellite and the intermediate satellite groups of 10 rats each were fasted overnight, blood and urine samples collected and were sacrificed for anatomic pathology evaluation including microscopic tissue evaluation.

 

Rats from the low target concentration group were exposed to a gravimetrically determined aerosol concentration of 0.0017 ± 0.00022 mg/L of the test substance (mean ± SD), which was characterized by a mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of 2.37 μm (2.72). Rats from the intermediate target concentration group were exposed to 0.012 ± 0.0039 mg/L of the test substance with a MMAD of 2.15 μm (2.77). Rats from the high target concentration group were exposed to 0.025 ± 0.0011 mg/L of the test substance with a MMAD of 2.12 μm (2.79). Rats from the intermediate satellite target concentration group were exposed to 0.012 ± 0.0013 mg/L of the test substance with a MMAD of 2.61 μm (2.84).  

Exposure to the test substance did not result in test substance related adverse changes in body weight, weekly feed and water consumption, and no clinical signs of toxicity were observed during the course of this study. There were no exposure-related differences in haematology or coagulation, clinical biochemistry parameters in male or female rats following the treatment or recovery periods.  

There were no test substance-related organ weight changes or gross pathological changes observed in this study. No test-substance related changes in microscopic histopathology were observed in this study. A modest number of animals (both sexes), including controls, had mild to marked retinal degeneration. Retinal degeneration was bilaterally distributed in a majority of the affected animals. The microscopic changes observed in the retina were not considered to be test substance related effects based on the following observations: Retinal lesions were observed in all groups (except at 0.001 mg/L), including controls, and in all rats in the control recovery/satellite groups in this study. In addition, in a previous 2 year chronic study with the test substance, no test substance related retinal changes were present at any dose in rats.  

Under the conditions of this study, the no-observable-adverse-effect-concentration (NOAEC) for the test substance was 0.025 mg/L for male and female rats.