Registration Dossier

Administrative data

Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Cross-referenceopen allclose all
Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 20, 2011 to February 07, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
- Storage condition of test material: Stored at room temperature and was considered stable under these conditions
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC.
- Age at study initiation: Approximately 7 weeks
- Weight at study initiation: 187-240 g for males; 149-184 g for females at randomization
- Housing: Animals were housed individually in clean, stainless steel, wire-mesh cages suspended above cage-board.
- Diet: Basal diet; PMI Nutrition International, LLC, Certified Rodent LabDiet® 5002 (meal), ad libitum
- Water: Reverse osmosis-treated (on-site) drinking water, ad libitum
- Food and water were withheld during each daily exposure period.
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature: 70.4-71.5 °F (21.3-21.9 °C)
- Humidity: 36.6-52.6 %
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: September 20, 2011 To: February 07, 2012
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 1.3 - <= 2.2 µm
Remarks on MMAD:
Mean MMAD: 1.4, 1.3 and 2.2 µm for 1, 10 and 100 mg/m3, respectively
Mean GSD: 3.59, 4.00 and 2.30 for 1, 10 and 100 mg/m3, respectively
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 7.9-L conventional nose-only exposure systems with synthetic rubber grommets in exposure ports to engage animal holding tubes.
- Method of holding animals in test chamber: To perform nose-only exposure, it is necessary to restrain the rats in specially designed nose-only holding tubes. The 6-hour period of restraint was necessary to achieve the exposure duration.
- Source and rate of air: HEPA- and charcoal-filtered air source (WIL Research Inhalation Department supply air source) or breathing-quality in-house compressed air source.
- Method of conditioning air: Charcoal- and HEPA-filters
- System of generating aerosols: Aerosolized test substance was generated using a Collison nebulizer.
- Temperature, humidity in air chamber: Mean temperature ranged from 20-21 °C and the mean relative humidity ranged from 43-56 %.
- Air flow rate: Daily mean ventilation rate ranged from 44.3-47.9 liters per minute (LPM).
- Method of particle size determination: Aerosol particle size determinations were conducted for each test substance exposure system using a 7-stage stainless steel cascade impactor.
- Treatment of exhaust air: All test substance atmosphere exposure system exhaust passed through a Solberg canister filter prior to entering the facility exhaust system, which consisted of redundant exhaust blowers proceeded by charcoal- and HEPA-filters.

TEST ATMOSPHERE
- Brief description of analytical method used:
Nominal exposure concentrations: Due to the small amount of test substance that was required for the study, a nominal concentration was not calculated.
Actual exposure concentrations: Actual aerosol mass concentrations of the test atmospheres for exposure systems 1, 3, and 4 (control, 10 and 100 mg/m3) were determined using standard gravimetric methods. Due to the volatility of the test material, standard gravimetric methods cannot be used for the analysis of the atmosphere in the Group 2 exposure system.
Analysed exposure concentrations: Analysed exposure concentrations of the total test material (combined vapour and aerosol) within exposure system 2 were determined at approximately 60-minute intervals using an appropriate gas chromatography (GC) method and were expressed as mg/m3.
- Samples taken from breathing zone: Yes
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A sample of the test substance was collected from prior to the initiation of exposure for purity and concentration analyses. Analyses of purity and stability were performed using a similar method from a sample collected at the end of the exposure period. All analyses were conducted using a gas chromatography method with flame ionization detection.
Results: Analyses conducted for a sample of the test substance collected prior to exposure initiation resulted in a purity of 90.8%. Following the completion of the exposure phase, another sample was collected that resulted in a purity of 89.3%. Comparison of these purity data indicated that the test substance was stable (98.3% of the initial test substance concentration after up to 34 days), based on the acceptance criteria for stability (i.e., the mean post-storage concentration was not <90% of the pre-storage value).
Mean exposure concentrations were 1.28, 9.8 and 98 mg/m3 for the target exposure concentrations 1, 10 and 100 mg/m3, respectively.
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
6 hours per day, 5 days per week for 2 weeks (10 total exposures)
Dose / conc.:
1 mg/m³ air (nominal)
Remarks:
0.15 ppm
Dose / conc.:
10 mg/m³ air (nominal)
Remarks:
1.5 ppm
Dose / conc.:
100 mg/m³ air (nominal)
Remarks:
14.9 ppm
No. of animals per sex per dose:
10
Control animals:
other: filtered air (control group)
Details on study design:
- Dose selection rationale: Prior to exposure of animals for the main study, preliminary animal exposures were conducted at 750 and then at 300 mg/m3 (89.4 and 44.7 ppm, respectively) for three days to assess tolerability of the test substance. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.
- Rationale for animal assignment: Animals judged suitable for assignment to the study were selected for use in a computerized randomization procedure based on body weight stratification in a block design.
Positive control:
None
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical examinations were performed daily, prior to exposure, during exposure (at the approximate midpoint for visible signs of animals in nose-only exposure restraint tubes), and 0 to 1 h following the end of exposure. The absence or presence of findings was recorded for individual animals at observations conducted prior to exposure and following the end of exposure. Only significant findings were recorded at the approximate midpoint of exposure observations. On non-exposure days, the animals were observed once daily.
- Survival: All animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity, except on the days of the scheduled necropsy.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were conducted on all animals during pretest prior to randomization, at the time of randomization prior to group assignment, and approximately weekly during the exposure period, including prior to the scheduled necropsy. Observations conducted prior to exposure were not performed on days when detailed physical examinations were conducted, provided they were conducted prior to
exposure.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded during the pretest period, at randomization, prior to the first exposure, and twice weekly during the exposure period (prior to the first and last exposure each 5-day exposure week) including the day before the first scheduled day of necropsy (nonfasted).
- Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights (fasted) were recorded on the day of the scheduled necropsy.

FOOD CONSUMPTION:
- Individual food consumption was recorded during the pretest period (prior to randomization) and twice weekly during the exposure period. Food intake was calculated as g/animal/day for the corresponding body weight intervals.

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected at the scheduled necropsy (study day 12).
- Anaesthetic used for blood collection: Yes; Blood was collected for haematology and standard serum chemistry evaluations via the retro-orbital sinus and for coagulation parameters via the vena cava from animals anesthetized by inhalation of isoflurane at the scheduled necropsy. (inhalation of isoflurane)
- Animals fasted: Yes; animals were fasted overnight prior to blood collection.
- How many animals: Blood samples for clinical pathology evaluations (haematology and serum chemistry) were collected from all animals, and blood samples for coagulation evaluations were collected from 5 animals/sex/group (Subgroup 1).
- Parameters checked:
HAEMATOLOGY AND COAGULATION: Total leukocyte count (WBC), Erythrocyte count (RBC), Haemoglobin (HGB), Haematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Platelet count (PLATELET), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Reticulocyte count Percent (RETIC), Absolute (RETIC ABSOLUTE), Mean Platelet Volume (MPV), Red cell distribution width (RDW), Haemoglobin Distribution Width (HDW), Differential leukocyte count - Percent and absolute (Neutrophil (NEU), Lymphocyte (LYMPH),Monocyte (MONO), Eosinophil (EOS), Basophil (BASO), Large unstained cell (LUC)), Platelet estimate, Red cell morphology (RBC Morphology)
CLINICAL CHEMISTRY: Albumin, Total protein, Globulin [by calculation], Albumin/globulin ratio (A/G Ratio), [by calculation], Total bilirubin (Total Bili) Urea nitrogen, Creatinine, Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma glutamyltransferase (GGT), Glucose, Total cholesterol (Cholesterol), Calcium, Chloride, Phosphorus, Potassium, Sodium, Triglycerides (Triglyceride), Sorbitol dehydrogenase (SDH) and Lactate dehydrogenase (LDH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; Animals were anesthetized by isoflurane inhalation and euthanized by exsanguination. For 5 animals/sex/group (BALF animals), a BAL was performed on the lungs as soon as possible after exsanguination and after completion of the BAL, all BALF animals were subjected to necropsy. The necropsies included, but were not limited to, examination of the external surface, all orifices, and the cranial, thoracic, abdominal, and pelvic cavities, including viscera. Tissues or gross lesions were not collected from the BALF animals (Subgroup 2).

HISTOPATHOLOGY: Yes; the following tissues and organs were collected from the histopathology animals (remaining 5 animals/sex/group; Subgroup 1) and placed in 10 % neutral-buffered formalin for examination:
Adrenals (2), Aorta, Bone with marrow, Femur (with joint)[a], Sternebrae, Bone marrow smear (from femur), Brain with olfactory blub - Cerebrum level 1, Cerebrum level 2 , Cerebellum with medulla/pons, Cervix, Epididymides (2)[b], Eyes with optic nerve (2) [c], Gastrointestinal tract, Oesophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Harderian glands (2), Heart, Kidneys (2), Lacrimal gland (exorbital [2]), Larynx, Liver (sections of 2 lobes), Lungs (including bronchi, fixed by constant pressure inflation with fixative), Lymph nodes - Auricular (2 [if visible]), Axillary (2), Bronchial (if visible), Mandibular (2), Mediastinal and bronchial (if visible), Mesenteric, Mammary glands (females only) [d], Nasal cavity (with turbinates) [e], Olfactory bulbs [e], Ovaries with oviducts (2) [f], Pancreas, Peripheral nerve (sciatic), Peyer’s patches, Pharynx, Pituitary, Prostate, Salivary glands (mandibular [2]), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin, Spinal cord (cervical, lumbar, thoracic), Spleen, Testes (2), Thymus, Thyroid (with parathyroids [2]) [f], Tongue, Trachea, Urinary bladder, Uterus, Vagina and Gross lesions

a = Bone marrow smears were obtained at scheduled necropsy from the femur, but not placed in formalin; slides were not examined.
b = Fixed in Bouin’s solution
c = Fixed in Davidson’s solution
d = A corresponding section of skin was taken from the same anatomical area for males.
e = Following the collection of the appropriate protocol-specified tissues, the entire head was removed and preserved (olfactory bulbs were severed from the brain and remained in the skull). Following decalcification, 6 cross-sections of the nasal cavities were prepared for microscopic examination in accordance with the method described by Morgan (1991) and Mery et al. (1994).
f = Oviducts and parathyroids were examined microscopically when in the plane of section and in all cases where a gross lesion was present.

After fixation, lungs with bronchi, bronchus associated lymphoid tissues [BALT], nasal tissues, nasal-associated lymphoid tissue [NALT], larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions) were trimmed. Trimmed tissues were processed into paraffin blocks, sectioned at 4 to 8 microns, mounted on glass microscope slides, and stained with haematoxylin and eosin. Microscopic examination of the lungs with bronchi, BALT, nasal tissues, NALT, larynx, trachea, mediastinal and bronchial lymph nodes, and gross lesions were performed for 5 animals/sex/group (Subgroup 1) not assigned for the BAL at the scheduled necropsy.

ORGAN WEIGHTS
The following organs were weighed from all animals at the scheduled necropsy:
Adrenals, Brain, Heart, Kidneys, Liver, Lungs (prior to inflation with fixative), Ovaries with oviducts, Spleen, Testes, Thymus, Thyroid with parathyroids*

Paired organs were weighed together. Designated (*) organs were weighed after fixation. Organ to final body weight and organ to brain weight ratios were calculated.
Other examinations:
Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation:
Prior to the scheduled necropsy, the animals were fasted overnight. At the time of necropsy, the 5 animals/sex/group selected for serum cytokine evaluations (Subgroup 2) were anesthetized by inhalation of isoflurane. Blood samples were collected from the vena cava into tubes containing no anticoagulant. A bronchoalveolar lavage (BAL) was performed on the lungs of 5 animals/sex/group (Subgroup 2) at the scheduled necropsy (study day 12).

BALF chemistry and cytology
The following parameters were evaluated:
Total and differential cell counts for: Alveolar macrophages, Neutrophils, Lymphocytes, Eosinophils, Basophils
Lactate dehydrogenase (LDH), Total protein, Alkaline Phosphatase

Serum and BALF cytokine evaluations
The following cytokines were evaluated:
TNF-α, IL-5, IL-10, ICAM-1, IFN-γ, IL-4, TGF-β, MCP-1, IL-1β, IL-13, MIP-2, RANTES

TNF = Tumour necrosis factor, IFN = Interferon, IL = Interleukin, TGF = Transforming growth factor, MIP = Macrophage inflammatory protein, ICAM = Intracellular adhesion molecule, MCP = Macrophage chemotactic protein, RANTES = Regulated upon activation, normal T-cell expressed, and presumably secreted.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Therefore, the use of reported individual values to calculate subsequent parameters or means will, in some instances, yield minor variations from those listed in the report data tables.

All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test substance-treated group to the control group by sex.

Body weight, body weight change, food consumption, clinical pathology data including BALF total protein, lactate dehydrogenase, alkaline phosphatase, and cytology parameters (with the exception of gamma glutamyltransferase), and organ weight data were subjected to a parametric one-way ANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the control group. Gamma glutamyltransferase values under range were assigned a value of 0.1 (half the lower limit of quantitation) for statistical analysis and reporting. Gamma glutamyltransferase data were subjected to the Kruskal-Wallis nonparametric ANOVA test (Kruskal and Wallis, 1952) to determine intergroup differences. If the ANOVA revealed significance (p<0.05), Dunn’s test (Dunn, 1964) was used to compare the test substance-treated groups to the control group.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no test substance-related clinical observations. All clinical findings in the test substance-treated groups were noted with similar incidence in the control group, were limited to single animals, were not noted in a dose-related manner and/or were common findings for laboratory rats of this age and strain.
Mortality:
no mortality observed
Description (incidence):
All animals survived to the scheduled necropsy
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on body weights.
- Transient lower body weight gains were present in the 100 mg/m3 group males and females during the exposure period, reaching statistical significance during study days 0 to 4 and/or 7 to 11. Statistically significantly lower cumulative body weight gains were recorded for the 100 mg/m3 group males from study day 0 to 11. Although lower cumulative body weight gains were noted in the 100 mg/m3 group females by the end of the study, the values were not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- There were no toxicologically significant effects on food consumption.
- Transient slightly lower food consumption was noted for the 100 mg/m3 group males and females during the exposure period. Although the differences were statistically significant compared to the control group from study days 0 to 4 and/or 7 to 11, these effects on food consumption were sporadic and not considered toxicologically relevant.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
There were no clear test substance-related differences in haematology parameters when compared to the control group. Reticulocyte counts were slightly lower in the 1, 10, and 100 mg/m3 group females and 100 mg/m3 group males when compared to the control group; however, a relationship with test substance administration was not established due to the lack of dose response in females.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no test substance-related differences in serum clinical chemistry parameters when compared to the control group. Alanine aminotransferase (ALT) was slightly lower in the 100 mg/m3 group males when compared to the control group; however, decreases in ALT have no known toxicological meaning, and this difference was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
- There were no test substance-related alterations in organ weights.
- Some organ weight differences were statistically significant when compared to the control group but were considered to be a result of a test substance-related effect on final body weight. For the 100 mg/m3 group males, mean adrenal gland weights (absolute and relative to brain weight) were lower, but the mean adrenal gland to body weight ratio was not statistically significantly lower. The mean final body weight was lower for this group, and this contributed to the lower adrenal gland weight values. Mean liver weights (absolute and relative to final body and brain weights) were also lower for the 100 mg/m3 group males, but these were affected by the lower final body weights in this male group. The mean liver weight relative to final body weight, which is a more appropriate permutation of the liver weight parameters, was very similar to the value in the 1 mg/m3 group indicating that there really were no differences in these relative weights. Adrenal gland, liver, and final body weights were not significantly altered in the 100 mg/m3 group females. Additionally, the adrenal gland weight values fell within the historical control reference ranges and there were no test substance-related microscopic changes in the livers of the 100 mg/m3 group males.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test substance-related macroscopic findings at the scheduled necropsy. All macroscopic findings noted were considered to be spontaneous and/or incidental in nature and unrelated to test substance exposure.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
- Test substance-related microscopic findings were present in the nasal cavity of males and females exposed to the test substance, but not in the larynx, trachea, lung, liver, or kidney. Nasal cavity changes tended to be more frequent in anterior portions of the nasal cavity (nasal levels II and III) and were also more frequent in males than in females.
Epithelial inflammation and degeneration were most prominent at nasal levels II (1, 10, and 100 mg/m3 group males and females) and III (1, 10, and 100 mg/m3 group males and 100 mg/m3 group females) and showed reduced incidence and severity in the posterior nasal levels (IV, V, and/or VI) in the 1, 10, and/or 100 mg/m3 group males and 10 and/or 100 mg/m3 group females.
- There were no other test substance-related histologic changes. The nasal-associated lymphoid tissue (NALT) and bronchial-associated lymphoid tissue (BALT) were unaffected by test substance exposure. Remaining histologic changes were considered to be incidental findings or related to some aspect of experimental manipulation other than exposure to the test substance. There was no test substance-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
LOAEL
Effect level:
1 mg/m³ air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
other: histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels. Therefore, a no-observed-effect level (NOEL) could not be determined for this study.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 mg/m³ air (nominal)
System:
respiratory system: upper respiratory tract
Organ:
nasal cavity
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes

None

Conclusions:
Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels. Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure levels.
Executive summary:

In a repeated dose toxicity study conducted according to the OECD Guideline 412 and in compliance with GLP, test material was administered by inhalation-aerosol to groups of Sprague-Dawley rats (10 animals/sex/dose) at the concentrations of 1, 10 and 100 mg/m3(0.15, 1.5, and 14.9 ppm, respectively) for 6 hours per day, 5 days per week for 2 weeks (10 total exposures). A concurrent control group was exposed to humidified, filtered air on a comparable regimen. Examinations during the study included: mortality, clinical observation, body weight change, food consumption, laboratory investigations: haematology, blood clinical chemistry, Bronchoalveolar lavage fluid (BALF) clinical pathology and serum and BALF cytokine evaluation, gross pathology, organ weights and histopathology.

 

Prior to the main study, a preliminary 3-day exposure (6 hours/day) was run at 750 and 300 mg/m3 (89.4 and 44.7 ppm, respectively) to allow selection of appropriate exposure concentrations. At 750 mg/m3, the animals were observed to have decreased body weights over the 3-day exposure period (-14 and -10% for males and females, respectively, compared to day 0) and significant clinical observations included rales and laboured respiration in 1/3 males and 2/3 females. At 300 mg/m3, the mean body weight was decreased by 8% and 3 % compared to the study day 0 values for males and females, respectively. At both concentrations, no animals died. Therefore, one third of the lowest concentration tested in the preliminary exposures, 100 mg/m3 (i.e.14.9 ppm), was chosen as the high exposure level for the main two-week study.

 

There were no test substance related effects on survival, clinical observations, haematology, coagulation, bronchoalveolar lavage clinical chemistry or cytology, serum cytokines, macroscopic findings, or organ weights. There were no toxicologically significant effects on body weight gain, food consumption, or BALF cytokine levels. The only test substance-related changes in BALF cytokine levels were the lower concentrations of sICAM in the 10 and 100 mg/m3group animals and lower concentrations of RANTES in the 100 mg/m3 group animals. No test substance-related microscopic findings were observed in the lungs, trachea, larynx, liver, or kidneys. Portal of entry effects suggestive of irritation were present in the nasal cavity of males and females exposed to test material. Epithelial inflammation and degeneration of the nasal cavity were noted in the 1, 10, and 100 mg/m3 group males and females. A dose-relationship was evident as higher incidences and severity of degeneration and subacute inflammation in males and females affecting transitional epithelium (nasal level II) at the 10 and 100 mg/m3 exposure levels, compared with effects at the 1 mg/m3 exposure. Based on severity and combined incidences for males and females, a similar test substance-relationship was evident for subacute inflammation of the respiratory epithelium at nasal level III. The findings were most prominent at nasal levels II and III and showed reduced incidence and severity in the posterior nasal levels (IV-VI), and were generally more frequent in males than in females at all exposure and nasal cavity levels. All nasal cavity findings were minimal to mild in severity and would be considered reversible with removal of the irritant. 

 

Results showed histologic changes indicative of localized irritation of the epithelia in the nasal cavity (upper airways) at all exposure levels up to 100 mg/m3. There were no test substance-related microscopic effects in the lower respiratory tract tissues (lungs, trachea, or larynx) or in the liver and kidneys, and there were no toxicologically significant effects on BALF cytokine levels.

 

Therefore a NOEL could not be determined for this study and the LOAEL is 1 mg/m3, based on the microscopic findings in the nasal tissues at all exposure level.

 

Based on the nasal irritation, test material is classified as H335: May cause respiratory irritation according to the Regulation (EC) No. 1272/2008 (CLP).

 

This study is considered as acceptable and satisfies the requirement for repeated dose inhalation toxicity endpoint.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance, with cis isomer (Z) as an impurity. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
Source and target substances have a common major constituent (trans-isomer) and the impurity of the target substance is the second major constituent of the source substance.
The studies were performed before the adoption of OECD TGs. However, the study design is similar to the one of the OECD TG 401 based on the exposure conditions and the key parameters assessed so the results are considered adequate and reliable for the purpose of prediction. The test material was not clearly identified but it is considered to represent the source substance in terms of constituents and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the acute oral toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VII, 8.5.1.
The study giving rise to the highest concern, i.e. the one giving the lowest value for the LD50 was used for the read-across. Therefore, there is no selection bias for study used for the prediction.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
0.5 mL/kg bw
Based on:
test mat.
Remarks on result:
other: Corresponding to 450 mg/kg bw
Mortality:
- Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively.
- No mortality was observed at 0.1 and 0.2 mL/kg bw.
Clinical signs:
Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semicomatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours.
The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours.
Body weight:
Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.
Gross pathology:
Autopsy of the mice that died revealed:
- Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver.
- Histological examination did not reveal liver necrosis.
Other findings:
None

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on the available data on the analogue substance, the registered substance is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.
Executive summary:

In an acute oral toxicity study, mice (1/sex/dose) were given a single oral (gavage) dose of test item at 0.1, 0.2, 0.5, 1.0 and 2.0 mL/kg bw. Animals were then observed for mortality, clinical signs and bodyweights for 7 days and were all sacrificed for macroscopic examination.

Mortality was observed in 1/2, 2/2 and 2/2 animals at 0.5, 1 and 2 mL/kg bw, respectively. No mortality was observed at 0.1 and 0.2 mL/kg bw. Most mice were showing signs of stress within 30 minutes-1 hour after treatment. The mice dosed at 1 and 2 mL/kg bw, and one animal dosed at 0.5 mL/kg bw, became semi-comatose/comatose, hypothermic and showed laboured breathing within 1 hour. All these animals died within 2-24 hours. The surviving animal dosed at 0.1 and 0.2 mL/kg bw recovered within 18 hours. Apart from the male mouse dosed at 0.5 mL/kg bw, all surviving animals gained weight during the 7 day observation period.

Autopsy of the mice that died revealed: Irritation of the stomach and small intestines, dark spleen, congested lungs and mottled liver. Histological examination did not reveal liver necrosis.

Mice oral LD50 = 0.5 mL/kg bw (corresponding to 541.5 mg/kg bw).

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw..

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity (cis- and trans-isomers).

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is the trans isomer (E), as a mono-constituent substance, with cis isomer (Z) as an impurity. The source substance is a reaction mass, composed of two diastereoisomers (the source substance [trans] and its cis-isomer).

3. ANALOGUE APPROACH JUSTIFICATION
Source and target substances have a common major constituent (trans-isomer) and the impurity of the target substance is the second major constituent of the source substance.
The studies were performed before the adoption of OECD TGs. However, the study design is similar to the one of the OECD TG 401 based on the exposure conditions and the key parameters assessed so the results are considered adequate and reliable for the purpose of prediction. The test material was not clearly identified but it is considered to represent the source substance in terms of constituents and impurities. The results of the studies are adequate for the purpose of classification and labelling.
Therefore, based on the considerations above, it can be concluded that the results of the acute oral toxicity studies conducted in both the rat and the mouse with the source substance are likely to predict the properties of the target substance and are considered as adequate to fulfil the information requirement of Annex VII, 8.5.1.
The study giving rise to the highest concern, i.e. the one giving the lowest value for the LD50 was used for the read-across. Therefore, there is no selection bias for study used for the prediction.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Sex:
male/female
Dose descriptor:
other: Rat LD50
Effect level:
1 560 mg/kg bw
Based on:
test mat.
95% CL:
1 290 - 1 880
Key result
Sex:
male/female
Dose descriptor:
other: Guinea pig LD50
Effect level:
1 410 mg/kg bw
Based on:
test mat.
95% CL:
1 130 - 1 780
Mortality:
Rats: Animals died within 1 hour to 4 days after treatment.
Guinea-pigs: Animals died within 3 to 6 days after treatment
Clinical signs:
Rats: Comatose soon after treatment, scrawny appearance for several days.
Guinea-pigs: Depression and coma
Body weight:
No data
Gross pathology:
No data

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Based on the available data on the analogue substance, the registered substance is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.
Executive summary:

In an acute oral toxicity study, rats (5/sex/dose) and guinea-pigs (number not reported) were administered the test substance by gavage with a stomach tube. Animals were then observed for mortality and clinical signs for 14 days.

In rats, coma was observed soon after treatment. Deaths occurred between 1 hour and 7 days and clinical signs were reported to be scrawny appearance and coma.

In guinea-pigs, deaths occurred between 3 and 6 days and clinical signs were reported to be depression and coma.

Rat oral LD50 = 1560 mg/kg bw (95% CL 1290-1880 mg/kg bw).

Guinea-pig oral LD50 = 1410 mg/kg bw (95% CL 1130 -1780 mg/kg bw).

 

Under the test conditions, test material is classified Category 4 (H302: Harmful if swallowed) according to the Regulation (EC) No. 1272/2008 and of the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw for rats and for guinea-pigs.

This study is considered as sufficiently reliable in a weight of evidence for the purpose o f acute oral toxicity endpoint.

Reason / purpose:
data waiving: supporting information
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 21, 1978 to April 23, 1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study was performed prior to the OECD test guideline No. 402 but the protocol is similar to that guidance.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
yes
Remarks:
(occlusive dressing, 3 animals/sex used; no details on environmental condition of animal room)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Test type:
standard acute method
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: C.S.E. colony, Branchville, N.J.
- Weight at study initiation: 2-3.4 kg
- Housing: Animals were housed individually under standard laboratory conditions.
- Diet: Purina Rabbit Chow, ad libitum
- Water: Water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS: Animals were housed and maintained according to 'Guide for the Care and use of Laboratory Animals'. DHEW Publication No. (NIH) 78-23. Revised 1978.

IN-LIFE DATES: From: November 11 1978 To: March 1979
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
TEST SITE
- Area of exposure: Dorsal surface of the trunk
- % coverage: 20 % of the entire body surface
- Type of wrap if used: Opened plastic sleeves (Baggies) were placed over the trunk and the posterior end was taped against the animal. Test material was applied over the prepared skin and the anterior end of the sleeve was taped against the animal, allowing the central portion to balloon. Elastic tape (Elastikon) was then lightly wrapped around the sleeve to reduce the chance of puncture or dislocation of the sleeve.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Excess material was cleansed from the skin using a clean disposable napkin moistened with saline.
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.8, 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw
Duration of exposure:
24 h
Doses:
0.8, 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw
No. of animals per sex per dose:
3
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for signs of toxicity or mortality twice daily (once daily on Saturday and Sunday) for 14 days.
- Necropsy of survivors performed: yes; any dead animals and all survivors were subjected to gross necropsy.
Statistics:
The LD50, slope and fiducial limits of the combined male and female mortality were estimated by the graphic method of Litchfield and Wilcoxon (1949)
Preliminary study:
Not applicable
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
1.77 mL/kg bw
Based on:
test mat.
95% CL:
> 1.35 - < 2.32
Remarks on result:
other: Equivalent to 1911.6 mg/kg bw (fiducial limits 1458-2506 mg/kg bw)
Mortality:
- No mortality was observed at 0.8 mL/kg bw.
- 1/6, 2/6, 4/6, 6/6 and 6/6 animals died at 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw, respectively. The majority of the deaths occurred within 24 hours.
Clinical signs:
- Systemic toxicity occurred in animals at all doses except 0.8 mL/kg bw.
- At 1.25 mL/kg bw, skin became erythematous and haemorrhagic areas were developed. Following drying and thickening, the skin was healed by termination.
- At 1.57 mL/kg bw, intradermal haemorrhage and eschar formation, with recovery toward normal skin texture in survivors were observed. Weakness was noted in all animals. Laboured breathing was observed in the first week.
- At 1.98 mL/kg bw, breathing was rapid and noisy and some animals had nasal discharge after 24 h. Respiratory symptoms persisted to death, but there was slow resolution in survivors. Haemorrhage and eschar formation were observed at the site of application.
- At 3.15 mL/kg bw, no meaningful clinical signs were observed before death of animals.
- At 5 mL/kg bw, slight erythema over application site was developed within 3 h and the skin became dark and haemorrhagic in 24 h. Nasal discharge was observed in animals which died.
Body weight:
- All surviving animals showed normal bodyweight gain over the 14-day study period, except for three males (one each from 0.8, 1.25 and 1.98 mL/kg bw group) and one female treated with 0.8 mL/kg bw which showed body weight loss.
Gross pathology:
- At 5 mL/kg bw effects observed at necropsy were congestion of the lungs.
- At 3.15 mL/kg bw, the major effects observed at necropsy were congestion of the lungs and failure of the vascular system characterized by haemorrhage, either locally into treated skin or into visceral organs
- At 1.98 mL/kg bw the major effects observed at necropsy were eschar formation and bruising of the skin and congestion of the lungs.
- At 1.57 and 1.25 mL/kg bw, 2/6 and 1/6 rabbits had congested lungs, respectively.
- At 0.8 mL/kg bw, no meaningful lesions were detected at necropsy.
Other findings:
None

None

Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
Under the test conditions, test material is classified Category 4 (H312: Harmful iin contact with skin) according to the Regulation (EC) No. 1272/2008 and to the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.
Executive summary:

In an acute dermal toxicity study, groups of New Zealand White rabbits (3/sex/dose) were given a single dermal application of undiluted test material at 0.8, 1.25,1.57, 1.98, 3.15 and 5.0 mL/kg bw to intact skin on the back. Test sites were covered with an occlusive dressing for 24 h. Animals were observed for mortality, clinical signs and bodyweights for 14 days. Animals which died and all survivors were subjected to gross necropsy.

No mortality was observed at 0.8 mL/kg bw. 1/6, 2/6, 4/6, 6/6 and 6/6 animals died at 1.25, 1.57, 1.98, 3.15 and 5 mL/kg bw, respectively. Thickening of the skin and varying degrees of erythema were observed. Eschar formation developed and necrotic patches began to exfoliate at about Day 7, continuing until termination, and leaving a healthy healing skin under the slough. Systemic toxicity occurred in animals at all doses except 0.8 mL/kg bw. Animals had dyspnea (either slow and laboured or very rapid breathing), nasal discharge (ranging from blood-tinged and frothy to thick and opaque) and congested lungs were found upon necropsy. The incidence and severity of the signs were least in the lower dose groups. All surviving animals showed normal bodyweight gain over the 14-day study period, except for three males (one each from 0.8, 1.25 and 1.98 mL/kg bw group) and one female treated with 0.8 mL/kg bw which showed body weight loss.

Dermal LD50 Combined = 1.77 mL/kg bw (fiducial limits1.35-2.32 mL/kg bw) [equivalent to 1911.6 mg/kg bw (fiducial limits 1458-2505.6 mg/kg bw).

Under the test conditions, test material is classified Category 4 (H312: Harmful iin contact with skin) according to the Regulation (EC) No. 1272/2008 and to the GHS as the LD50 value is comprised between 300 and 2000 mg/kg bw.

This study is considered as acceptable and satisfies the requirement for acute dermal toxicity endpoint.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion