Registration Dossier
Registration Dossier
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EC number: 939-368-0 | CAS number: -
Endpoint summary
Administrative data
Description of key information
OECD 413 study in rat (90-day inhalation toxicity study): NOAEC = 0.004 mg/L air.
OECD 422 study in rat (oral route): NOAEL (parental) = 133 mg/kg bw/day.
Key value for chemical safety assessment
Repeated dose toxicity: via oral route - systemic effects
Link to relevant study records
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- from 20 April 2012 to 5 December 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Reason / purpose for cross-reference:
- reference to other study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- Principles of method if other than guideline:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- N° 2011/40
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source:Charles River Laboratories France, L’Arbresle, France. Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free, (COBS-VAF®).
- Age at study initiation: the males were approximately 10 weeks old, the females were approximately 9 weeks old
- Weight at study initiation: males: a mean body weight of 415 g (range 381 g to 440 g), and female a mean body weight of 243 g (range: 218 g to 288 g).
- Fasting period before study: no
- Housing: The animals were individually housed, except during pairing, in polycarbonate cages (Techniplast 2154.940 cm²) with stainless steel lids, and containing autoclaved sawdust (SICSA, Alfortville, France).
Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period.
half hut was given as enrichment of the environment of the rats.
The cages were placed in numerical order on the racks.
- Diet (e.g. ad libitum): The animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 5776559 and 2626975 (SSNIFF Spezialdiäten GmbH, Soest, Germany) which was distributed weekly.
- Water (e.g. ad libitum): The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter)
- Acclimation period: the animals were acclimated for a period of 5 days before the beginning of the treatment period.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)
IN-LIFE DATES: From:2012-05-02 To: 2012-06-08 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Remarks:
- treated by reverse osmosis using an ELIX 5 apparatus
- Details on oral exposure:
- PPREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was mixed with the required quantity of vehicle.
The dosage formulations were prepared on a daily basis.
The dosage forms were stored and delivered at room temperature in brown flasks.
VEHICLE:
drinking water treated by reverse osmosis using an ELIX 5 apparatus
- Concentration in vehicle: 0, 30, 100 and 300 mg/kg of active ingredient
- Amount of vehicle (if gavage): 5 mL/kg - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda in dosage form samples was validated at CITixLAB prior to dosage form analysis.
The analytical method was validated for concentration ranging from 2 to 200 mg/mL.
The concentration of the test item in samples of each control and test item dosage form prepared for use in weeks 1, 3 and 5 was determined. In addition, the concentration of the test item in a sample of the high-dose test item form prepared for use in week 2 was determined.
Acceptance criterion: measured concentration = nominal concentration ± 10%
The validation of the analytical method was conducted in CIT/Study No. 38263 VAA and precise details concerning the checked parameters, acceptance criteria and obtained results were documented in the corresponding validation report.
Each analytical sequence consisted of at least:
+ A blank sample (diluent only),
+ Ten standard samples at nominal concentration, prepared from two independent standard solutions,
+ Study samples prepared from aliquots of the dosage forms.
The standard samples bracketed the dosage form samples.
The blank sample was checked for the absence of chromatographic interference. - Duration of treatment / exposure:
- The dosage forms were administered daily according to the following schedule:
. in the males:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (from study day 15 until study day 16 to 25),
- until sacrifice (at least 5 weeks in total)(from study day 17 to 26 until study day 36).
. in the females:
- 2 weeks before pairing (from study day 1 to 14),
- during the pairing period (from study day 15 to 25),
- during gestation (from study day 16 to 26 until study day 36 to 46),
- during lactation until day 5 post-partum inclusive (from study day 37 to 47 until study day 42 to 52),
Study day 1 corresponds to the first day of the treatment period. - Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
30; 100; 300 mg/kg bw/d
Basis:
other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri) - Remarks:
- Doses / Concentrations:
39.8; 133 and 398 mg/kg bw/d
Basis:
other: expressed as registered substance (UVCB 100%) - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The dose-levels were selected in agreement with the Sponsor based on a 2 week dose range finding study by oral route (gavage) in rat (CiToxLAB France/Study No. 38818 TSR). In this preliminary study, the following findings were recorded:
. mortality: at 1000 mg/kg/day, all animals were found dead or sacrificed moribund on study day 2. Severe clinical signs preceded the deaths or decision to sacrifice (i.e.: thin appearance, hunched posture, piloerection, loud breathing, soiled fur, cold to the touch and/or hypoactivity),
. clinical signs: all test item-treated animals had ptyalism (at the end of the treatment period at 100 or 300 mg/kg/day). Clinical signs (thin appearance, hunched posture, loud breathing, and/or dyspnea) were recorded at 100 or 300 mg/kg/day, but with an overall increased incidence in males when compared to females,
. body weight: there were no effects on mean body weight in all surviving animals at 100 or 300 mg/kg/day,
. food consumption: there were no effects on mean food consumption in all surviving animals at 100 or 300 mg/kg/day,
. pathology: in found dead or sacrificed moribund animals, there were red discoloration(s) and/or thickened wall in stomach, jejunum, ileum, cecum, forestomach and/or duodenum. The digestive organs were or not distended with gas. In surviving males, there were increased (≥ 10% vs. controls) absolute and/or relative liver, kidneys and testes weights at 300 mg/kg/day. In surviving females, there were increased (≥ 10% vs. controls) absolute and/or relative kidneys weight at 300 mg/kg/day and decreased (≤ -8 to -15% vs. controls) absolute and/or relative heart and ovaries weight from 100 mg/kg/day. At necropsy there were a few isolated findings (liver with yellow discoloration at 100 mg/kg/day in 1/3 males and lung(s) with dilatation in 1/3 females at 300 mg/kg/day).
Overall, 1000 mg/kg/day was considered to be an excessive high dose-level for the further OECD 422 study while 300 mg/kg/day could be an appropriate high dose-level.
Therefore 300 mg/kg/day was selected as the high dose-level. The low-dose and mid dose have been selected using a ratio representing a 3-fold interval (i.e. 30 and 100 mg/kg/day). - Positive control:
- not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day before the treatment period and at least twice a day during the treatment period
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical examinations were performed on all animals outside the home cage, in a standard arena, once before the beginning of the treatment period and then once a week until the end of the study.
BODY WEIGHT: Yes
- Time schedule for examinations:
+ males: on the first day of treatment (day 1), then once a week until sacrifice.
+ femelles: on the first day of treatment (day 1), then once a week until mated and on days 0, 7, 14 and 20 p.c. and days 1 and 5 p.p..
The body weight of female sacrificed on day 25 p.c. for no delivery was recorded the day of sacrifice, presented in the report but not including in the statistical evaluation.
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
The quantity of food consumed by each male was measured once a week, over a 7 day period, from the first day of treatment until the start of the pairing period.
The quantity of food consumed by each female was measured once a week, over a 7 day period, from the first day of treatment until the start of pairing period, during pregnancy at the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for interval days 1 5 p.p..
During the pairing period, the food consumption was measured neither for males nor females.
Food intake per animal and per day was calculated by noting the difference between the food given and that in the food-hopper the next time.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: Yes pupillary reflex during the FOB test
- Dose groups that were examined: 5 males and 5 female per group
HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table 7.5.1/1 were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes light isoflurane anesthesia
- Animals fasted: Yes for an overnight period of at least 14 hours
- How many animals: the first five males and the first five females to deliver from each group
- Parameters checked in table 7.5.1/1 were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once at the end of the treatment period
- Dose groups that were examined: The first five males and the first five females to deliver
- Battery of functions tested:
+"touch escape" or ease of removal from the cage
+In the hand: fur appearance, salivation, lachrymation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis)
+ In the standard arena (2-minute recording): grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper-activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia
+ The following parameter measurements, reflexes and responses were recorded:
touch response,
forelimb grip strength,
pupillary reflex,
visual stimulus response,
auditory startle reflex,
tail pinch response,
righting reflex,
landing foot splay,
at the end of observation: rectal temperature.
OTHER:
+ at the end of observation period: rectal temperature.
+Motor activity of all animals was measured once by automated infra-red sensor equipment over a 60-minute period - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes (see table 7.5.1/2)
HISTOPATHOLOGY: Yes (see table 7.5.1/2) - Other examinations:
- no other examination
- Statistics:
- Citox software (version D.06) was used for the statistical analyses of hematology and blood biochemistry data according to the sequence, described in attached document.
Other data was compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01) according to the sequence described in the attached document. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- There were no unscheduled deaths in controls or test item-treated groups. Emaciated appearance from 100 mg/kg bw/d in few males. Hypoactivity and loud/abdominal breathing at 300 mg/kg bw/d in some females. Ptyalism, observed in most high dose animals, was considered to be related to the test item but of minor toxicological importance.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- There were no unscheduled deaths in controls or test item-treated groups. Emaciated appearance from 100 mg/kg bw/d in few males. Hypoactivity and loud/abdominal breathing at 300 mg/kg bw/d in some females. Ptyalism, observed in most high dose animals, was considered to be related to the test item but of minor toxicological importance.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant decreases in body weight changes from 100 mg/kg bw/d, resulting in body weight loss at 300 mg/kg bw/d in males. In females, no effect on body weight was observed.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Statistically significant decrease in mean food consumption in males given 300 mg/kg bw/d for the period of days 1 to 8. No effect on food consumption in females.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- Minimal increase in adrenal weights in females given the test item at 300 mg/kg bw/d. In absence of microscopic correlates, this variation was considered to be of equivocal significance.
- Gross pathological findings:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Details on results:
- no further details
- Dose descriptor:
- NOAEL
- Effect level:
- 100 mg/kg bw/day (nominal)
- Based on:
- other: expressed as active material in mg/kg bw/d (purity 75.4% considering the three forms of sodium isopropylnaphtalenesulphonate: mono, di and tri)
- Sex:
- male
- Basis for effect level:
- other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change
- Dose descriptor:
- NOAEL
- Effect level:
- 133 mg/kg bw/day (nominal)
- Based on:
- other: expressed as registered substance (UVCB 100%)
- Sex:
- male
- Basis for effect level:
- other: Clinical signs in males at 100 mg/kg bw/d: emaciated appearance, decrease in body weight change
- Critical effects observed:
- not specified
- Conclusions:
- Under the test conditions, the test item Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda induced slight clinical signs in males only from the dose of 100 mg/kg bw/d, but no adverse systemic effects were observed after a repeated dose exposure to the test item. The NOAEL was determined to be 100 mg/kg bw/d when expressed as the active material and 133 mg/kg bw/d when expressed as the registered substance (UVCB at 100%) based on clinical signs observed in male rats. Therefore no classification as STOT-RE is required according to the Regulation (EC) 1272/2008 (CLP) and as harmful after repeated dose exposure according to the Directive 67/548/EEC.
- Executive summary:
In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test by oral route performed in compliance with the GLP and in accordance with the OECD 422 test guideline, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda (purity of 75.4%) diluted in water was administered daily by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 0; 30; 100 and 300 mg/kg bw/d (based on the three forms of sodium isopropylnaphtalenesulphonate (mono, di and tri) content i.e. 0; 39.8; 133 and 398 mg/kg bw/d in terms of registered substance). Males were exposed 2 weeks before pairing, during pairing and until sacrifice (at least 5 weeks in total) whereas females were exposed 2 weeks before pairing, during pairing, during gestation and during lactation until day 5 post-partum (6-7 weeks in total).
Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. Body weight was recorded weekly until sacrifice and then at designated intervals throughout gestation and lactation for the females. A Functional Observation Battery was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology and blood biochemistry parameters. The males were sacrificed after completion of the mating period. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups. Dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.
The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 5 remained within an acceptable range of -3.7% to +4.4 when compared to the nominal values. SUPRAGIL WP was not detected in control samples.
With regards to repeated-dose toxicity parameters, the following observations were made:
There were no unscheduled deaths in control or test item-treated groups. Emaciated appearance was observed in few males from 100 mg/kg bw/d. Hypoactivity and loud/abdominal breathing was observed in some females exposed to 300 mg/kg bw/d. Statistically significant decrease in body weight changes were observed in males from 100 mg/kg bw/d for the period of days 1 to 8 only, resulting in body weight loss at 300 mg/kg bw/d. This was associated with a statistically significant decrease in mean food consumption at 300 mg/kg bw/d for the period of days 1 to 8. In females, there was neither effect on body weight nor on food consumption. A minimal increase in adrenal weights was observed in females given the test item at 300 mg/kg bw/d. But in absence of microscopic correlates, this variation was considered to be of equivocal significance.
Based on the results of this study, 133 mg/kg bw/d of test item (as registered) was established as the No Observed Adverse Effect Level (NOAEL) considering the clinical signs observed in males.
Therefore, the registered substance is not classified for repeated dose toxicity according to the classification criteria of the Regulation (EC) 1272/2008 (CLP) and of the Directive 67/548/EEC. This study is considered as acceptable as it satisfied the criteria of the OECD Guideline No. 422.
Reference
no other information
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEL
- 133 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- GLP and OECD 422 compliant study (Klimisch 1)
- System:
- other: clinical signs
Repeated dose toxicity: inhalation - systemic effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 25 June 2017 to 23 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks old at the initiation of treatment
- Weight at study initiation: Males: 221-250 g; females: 156-173 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 (males) - 24 (females) days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 27.9°C
- Humidity (%): 30 - 78 %
- Air changes (per hr): 15-20 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 25 June to 30 September 2017 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.68 - <= 1.16 µm
- Geometric standard deviation (GSD):
- 2.08
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.68 (GSD = 2.03)
Group 3 (Mid dose): MMAD = 0.99 (GSD = 2.08)
Group 2 (High dose): MMAD = 1.16 (GSD = 1.89) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber was used. The rate of formulation use was controlled by a syringe pump. Verification of the target concentration, particle size and particle distribution was measured gravimetrically. Technical trials included filter analysis. Technical trials with the aqueous formulations had shown that the droplets become a powder in the dry air, so the rats were exposed principally to a powder (samples collected at the animal breathing zone were used for atmosphere characterisation).
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 13 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.004 mg/L air (nominal)
- Dose / conc.:
- 0.01 mg/L air (nominal)
- Dose / conc.:
- 0.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The dose levels were selected based on previous data available, including the results of preliminary dose range finding studies in the rat
(see 4-week studies 15/352-212PE and 15/352-212PER dated 2017-218 reported in section 7.5.2).
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. As a minimumon exposure days, individual, clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observation was performed at least twice (as
soon as practicable after removal from restraint, and approximately one hour after completion of the exposure). In non-treatment days, clinical observation was made once.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena. The first detailed observation was made on Day 8 then once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, and twice a week thereafter (except Week 1) including Day 90 (last exposure day) and fasted on Day 91.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was conducted in all animals before treatment and on Week 13.
Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL ciclopentolato hydrochloride; Batch No.: 160515, exp.: May 2021; Batch No.: 170120, exp.: January 2022) into the conjunctival sac. The evaluation was performed by external examination and using a Gowllands or Heine Omega 500 ophthalmoscope.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
Towards the end of the treatment period on Day 77-78, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment, on one of the 2 days per week when there was no exposure.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were also painted and measured, but they were not evaluated.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively
rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed
to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day.
The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from each dose and control group were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.
IMMUNOLOGY: No - Sacrifice and pathology:
- Gross necropsy was performed on each animal. Terminally on Day 91 surviving animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- ORGAN WEIGHTS: (see table 3)
EXAMINATION OF VAGINAL SMEARS:
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution.
The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues. - Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chisquared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment related clinical signs were observed in any group.
Alopecia or fur thin were commonly recorded in all groups, transient, sporadic diuresis was recorded in males on Days 19, 20 and 23 and in females on 12, 13 and 16 in the High dose group. Sporadic appearance of scars was also observed in Low and High Dose male groups. These findings were not considered to be adverse effects of treatment. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control groups, there were significantly lower body weights at the end of the treatment in the Mid and High doses (9.4% and 13.1% (p<0.01) in Mid and High dose males and 5.2% (p<0.05) in High dose females) and the overall body weight gains were significantly lower in both sexes (27.3% and 37.9% in Mid and High dose males; 16.0% and 19.5% in Mid and High dose females).
It is considered that the reduced weight gain in Mid and High dose males was an adverse effect of treatment. Similarly, but to a lesser extent, High dose females were also affected; Mid dose female weight differences were similar to the High dose females, but were generally not statistically different from controls. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food intakes reflected the body weight gain differences, with lower food intake in the High dose males most weeks and some weeks in the Mid dose males, with the overall
food intake significantly lower than control only in High dose males. There were no statistical differences in females. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. All examined animals were found to be normal.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the haematology parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is considered that there were
no adverse haematology changes in the study.
The WBC count for some control males was higher than typically seen; apparently statistically lower WBC values in the treated groups was not considered to be a
treatment effect, taking into account control data from a contemporaneous study. This difference consequentially caused the absolute WBC sub-populations to be statistically
different as well, but since the relative counts were clearly not affected by treatment, these differences were not of biological significance. There were no effects on female
WBC parameters.
Small differences in RBC, Hct, MCV or MCH were all close to the expected range without any consistent indication of a test item effect on erythrocyte parameters, these
were not considered to reflect an adverse effect of treatment. Similarly, the differences in APTT or PTT were relatively small and were similarly considered as sporadic
statistical differences and not an adverse effect of treatment.
None of the statistical differences in haematological parameters are considered to represent an adverse effect of the test item. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the clinical chemistry parameters, there were a few statistical differences. To establish their relationship with treatment, control data from a contemporaneous study and the data from the clinical chemistry of the preliminary study with this test item were examined. The statistical differences for Creatin were clearly without a dose response.
The statistical differences for Sodium, ALT and ALKP were close to the expected control range, did not show indications of a dose response and showed no differences in the preliminary 28-day study (Report 15/353-212PE) hence these statistical differences were not considered to represent a treatment related effect. The lower Glucose and Triglycerides in High dose males, and the apparent reductions in Protein Albumin and A/G ratio in High dose females were all changes that mirrored the changes seen in the 28-day preliminary study, hence although the magnitude of the changes were not great, they were ascribed to being an effect of treatment. In the absence of any histological changes in the liver or kidney, the toxicological significance of these changes is equivocal; the magnitude of the differences suggests these are not indicative of significant adverse systemic toxicity. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity in any dose group.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The following treatment related changes were observed in the mean organ weights:
The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females. The lung weight relative to body weight was about 21% and 55% above control in Mid and High dose males respectively; in female Mid and High dose groups the differences were similar at about 24% and 58% respectively.
In the thymus (an organ generally sensitive to stress) there appeared to be a degree of individual stress related with treatment. The absolute and relative (to body and/or brain) thymus weights were statistically significantly lower in the Mid and High dose groups of males (abs. weights: 29.7% and 35.2% (p<0.01); rel. to bodyweights: 22.8% and 25.6% (p<0.01) and rel. to brain: 26.8% and 32.9% (p<0.01)) and in the High dose group of the females (abs. weight: 25.5% (p<0.05); rel. to brain: 26.8% (p<0.05)). Histopathology confirmed decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
Other statistical differences in organ weight were generally directly associated with the body weight differences, with lower absolute weights or higher relative weights of some organs, but with no relationship with systemic effects on the organs. The female adrenal (absolute and relative) appeared higher than control, but in the absence of any histopathological change this difference was not considered to be an adverse effect of treatment. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Test item-related macroscopic changes were not seen during the necropsy.
Other findings, like thin fur, scares on the skin, enlarged mediastinal lymph node (in 1/10 High dose male), dark red discoloration of the thymus (in 1/10 Low dose male) and the dilatation of uterus could be considered as background or procedure related. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium were microscopically observed in the lungs from the Mid and High dose
male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). There were no effects observed in the Low dose. The histological changes are correlated with organ weight changes. This histological change is considered to reflect a local irritation response caused by the presence of the test item in the atmosphere; the effect is considered to be an adverse effect. There were no other changes suggestive of a degenerative pathology.
Histopathology of the thymus showed decrease of size/cellularity of the thymic cortex in High dose males. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
The decreased cellularity in the thymus and other sporadic changes, like increased cellularity in the mediastinal lymph node, inflammatory cell infiltrate in the prostate,congestion/haemorrhage in the thymus, erosion/ulceration in the skin and signs of oestrus were considered as incidental or background. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Examination of vaginal smeares: There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 0.004 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.01 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused decreased
body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item.
There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m3). - Executive summary:
The objective of this 90-day study was to evaluate the potential toxic effects of the test item, Supragil WP, when administered to Wistar (Han) rats via inhalation route for at least 90 days. This study was carried out according to OECD test guideline No. 413 (September 2009), in compliance with the Good Laboratory Practices (GLP).
Three groups of 10 male and 10 female Wistar rats Crl:WI (Han) were exposed to the test item, at target concentrations of 0.004, 0.01 and 0.04 mg/L using a nose-only
exposure system. A vehicle control group of 10 male and 10 female rats was exposed to distilled water. The animals were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). Start of the exposure for all 4 groups was Day 1.
The treatment was performed in 4 inhalation towers in parallel, one tower for each treatment groups and an additional tower for the vehicle control group.
Surviving animals including controls were terminated on the day following the last exposure on Day 91.
The achieved concentrations and particle size distributions for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.
Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.
Results:
There was no mortality in the study.
No treatment related clinical signs were observed in the treatment groups.
There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity; hence no neurological effects were identified in any dose group.
There were significantly lower body weights at the end of the treatment in the Mid and High dose groups and the body weight gains were significantly lower in both sexes, particularly in males. Food intakes reflected the body weight gain differences.
No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination.
In the haematology and clinical chemistry parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is
considered that there were no adverse clinical pathology changes in the study.
There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the
oestrus phases.
The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females.
These changes correlated with a local irritation response of hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium observed microscopically in the lungs from the Mid and High dose male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). These changes were considered to be adverse effects of the test item.
An apparent stress effect was seen in thymus weights on some Mid and High dose males correlated, at histology, with a decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
There were no other changes in organ weights, necropsy or histological observations.
In summary, Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused
decreased body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item. There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m3).
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 28 December 2016 to 01 February 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old at the initiation of treatment
- Weight at study initiation: Males: 250-282 g; females: 172-191 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 19 days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 23.5°C
- Humidity (%): 24 - 61 %
- Air changes (per hr): At least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 28 December 2016 To: 23 January 2017 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.63 - <= 1.44 µm
- Geometric standard deviation (GSD):
- 2.62
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.63 (GSD = 2.62)
Group 3 (Mid dose): MMAD = 1.05 (GSD = 2.34)
Group 2 (High dose): MMAD = 1.44 (GSD = 1.82) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber was used. The rate of formulation use was controlled by a syringe pump. Verification of the target concentration, particle size and particle distribution was measured gravimetrically. Technical trials included filter analysis. Technical trials with the aqueous formulations had shown that the droplets become a powder in the dry air, so the rats were exposed principally to a powder (samples
collected at the animal breathing zone were used for atmosphere characterisation).
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Air change rate:
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 4 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.004 mg/L air (nominal)
- Dose / conc.:
- 0.01 mg/L air (nominal)
- Dose / conc.:
- 0.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The concentration levels were determined based on the available experimental data from a preliminary 5 days escalating dose study and a previous 28-day DRF study (study 15/352-212PE dated 2017 reported in section 7.5.2).
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after
removal from restraint, and approximately one hour after completion of the exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal were recorded with precision of 1 g on Day 1 (before the exposure), once a week thereafter and on Days 26 and 27 (prior to necropsy, fasted weight).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All animals were euthanized under pentobarbital anaesthesia on Day 27 (RELEASE, Lot No.: 106075; Expiry: July 2018; WDT, Produced by Garbsen, Germany) by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was
observed macroscopically. All abnormalities were recorded with details of the location, colour, shape and size, as appropriate.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- Organ weights: (see table 3)
- Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No clinical sign was recorded in the Control group. Slight laboured respiration was observed in all treatment groups in some treatment days in each animal during and right after the exposure. The frequency of this observation was increased with the dose. In one High dose male decreased activity (slight) was recorded on Day 3.
Red-brown staining, wet fur, alopecia or scar were also recorded. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. - Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant differences in body weight in any group. The overall body weight gain was not statistically different to controls; although the mean values were 48% and 38% below control in the High dose males and females respectively, and at the Mid dose about 25% lower in both sexes.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food intakes reflected the body weight gain differences.
- Food efficiency:
- not examined
- 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 toxicologically significant effects in investigated haematology parameters between test item treated animals and the controls.
Occasionally, in the haematology parameters few statistical differences were seen (e.g., decreased haemoglobin, mean cell haemoglobin in the High dose males, decreased white blood cell, mean cell haemoglobin, platelet count and lymphocytes in Mid dose females). All groups were within the expected historical control range and there were no coherent trends, these statistical differences were considered to be incidental.
There was no significant change in the blood clotting parameters. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- In both sexes statistically significant differences were seen in electrolytes, protein or albumin, but most were without a clear dose-response or were in the normal range. There were no differences that were clearly related to treatment.
- 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):
- When compared to the controls, the following changes were observed in the mean organ weights:
The absolute and relative (to body and brain) lung weights were statistically significantly higher (p<0.01) in High dose group of both sexes, the relative lung weight to body weight was also higher in the male Mid dose group (p<0.05). - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related findings were identified in the lungs from the male High dose group only. Minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats. These changes were considered to be treatment related and are in line with the lung organ weight differences observed.
Other changes, like multifocal, mononuclear cell infiltrate in the liver of 1/10 Mid dose male and signs of oestrus in 1/5 Mid and 2/5 High dose females were considered as incidental or background. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 0.004 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.01 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The exposure to the test item Supragil WP in distilled water to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels 0.004 to 0.04 mg/L caused transient slight laboured respiration and noisy respiration.
There were no statistically significant effects on body weight or clinical pathology. Lung weights were significantly increased in both sexes at the High dose, with slightly higher relative weights in Mid dose males. Histologically, minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
These changes in the High Dose were similar to those observed in the Low Dose of the previous DRF (15/352-212PE).
The NOAEL is considered to be the Low dose (0.004 mg/L) although the changes in the Mid dose (0.01 mg/L) were minimal with no histopathological effects observed. Dose levels above the High dose (0.04 mg/L) are considered to be too high for a 90 day study.
Based on the results of the two 28d DRF studies, the dose levels for the subsequent 90d GLP study were set at 0.04, 0.01 and 0.004 mg/L. - Executive summary:
The objective of this additional 4-week dose range finding study is to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item when administered to Wistar rats via inhalation route for 4 weeks with the aim of inducing toxic effects but no death or suffering at the highest
concentration, and a NOAEL at the lowest concentration level. In a previous 28-day dose range finding (DRF) study (No. 15/352-212PE, Ref. 8) the NOAEL was not identified. The results of the study would be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.
Five male and 5 female Wistar rats Crl:WI (Han) in each group were exposed, by a 6 hour nose-only exposure for consecutive 5 days/week, to the following target
aerosol concentrations of the test item: 0.004, 0.01 and 0.04 mg/L. A control group of 5 male and 5 female rats was exposed to filtered air. The duration of the study was 4 weeks, each week consisted of 5 consecutive treatment days and 2 days without treatment. The total number of exposures for each animal was 20. The animals were sacrificed on the day following the last exposure in Week 4.
In the previous 28-day DRF study where 0.2 mg/L, 0.09 mg/L and 0.04 mg/L were tested, significant toxicity was observed in each dose groups and none of these
concentrations was low enough to determine the NOAEL. Therefore, in the current study, the low dose (0.04 mg/L) of the previous study was selected as high dose, and two lower concentrations were tested to find the non-toxic level.
The animals were exposed to the test atmosphere with formulations made in distilled water using a nose-only exposure system. It was not technically feasible to produce the low concentrations using the milled batch as a powder. The test item was generated using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) from 0.048 % (w/w), 0.12 % (w/w) and 0.48 % (w/w) formulations for the Low, Mid and High Doses, respectively. Analytically measured concentrations of 0.004 mg/L, 0.0126 mg/L and 0.0378 mg/L were achieved in the respective groups, the target concentrations being 0.004 mg/L, 0.01 mg/L and 0.04 mg/L. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group. Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry). Gross macroscopic examination was performed at necropsy and selected organs were weighed.
Full histopathology of the whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) was performed in each group.
The test atmosphere concentration was monitored based on the gravimetric and chemical analysis. The results of the test atmosphere characterization were
considered suitable for the study purposes.
Results
No mortality was observed in the study.
Slight transient laboured respiration was observed in all dose groups during or after the treatment, more frequently in the higher doses.
The body weight gain in the High Dose in both sexes was apparently lower than control, but without statistical significance. The same trend was seen at the Mid dose. There were no treatment related changes in clinical pathology. When compared to the controls, the following changes were observed in the mean
organ weights: Lung weights were statistically significantly higher (p<0.01) for absolute and relative weights in the High dose group in both sexes. The relative lung to body weight in male Mid dose group were also higher (p<0.05).
Test item-related macroscopic changes were not seen during the necropsy. Minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar
epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
Conclusion:
The exposure to the test item Supragil WP in distilled water to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels 0.004 to
0.04 mg/L caused transient slight laboured respiration and noisy respiration. There were no statistically significant effects on body weight or clinical pathology.
Lung weights were significantly increased in both sexes at the High dose, with slightly higher relative weights in Mid dose males. Histologically, minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
These changes in the High Dose were similar to those observed in the Low Dose of the previous DRF (15/352-212PE).
The NOAEL is considered to be the Low dose (0.004 mg/L) although the changes in the Mid dose (0.01 mg/L) were minimal with no histopathological effects
observed. Dose levels above the High dose (0.04 mg/L) are considered to be too high for a 90 day study.
Based on the results of the two 28d DRF studies, the dose levels for the subsequent 90d GLP study were set at 0.04, 0.01 and 0.004 mg/L.
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 19 March 2016 to 06 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old at the initiation of treatment
- Weight at study initiation: Males: 194-226 g; females: 155-184 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 – 24.3°C
- Humidity (%): 32 - 59 %
- Air changes (per hr): At least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 19 March 2016 To: 14 April 2016 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.91 - <= 1.19 µm
- Geometric standard deviation (GSD):
- 3.49
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.91 (GSD = 3.16)
Group 3 (Mid dose): MMAD = 1.19 (GSD = 3.49)
Group 2 (High dose): MMAD = 1.08 (GSD = 2.87) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and
constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, Palas RBG1000 (Palas GmbH, Karlsruhe, Germany) rotating brush generator were used connected to pressurized air supply. Atmosphere generation was dynamic. Fresh test atmosphere from the generation system was constantly supplied to the inner plenum (distribution chamber) of the
exposure system from where was distributed to the individual exposure ports.
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Air change rate:
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 4 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.04 mg/L air (nominal)
- Dose / conc.:
- 0.09 mg/L air (nominal)
- Dose / conc.:
- 0.2 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The concentration levels were determined based on the available experimental data from a preliminary 5 days escalating dose study.
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after
removal from restraint, and approximately one hour after completion of the exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal were recorded with precision of 1 g on Day 1 (before the exposure), once a week thereafter and on Days 26 and 27 (prior to necropsy, fasted weight).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All animals were euthanised under pentobarbital anaesthesia (Euthanimal 40%; Lot No.: 1409236-06; Expiry: 09-2017; Produced by Alfasan Nederland BV, Kulpersweg 9, Woerden, Netherlands) by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. All abnormalities were recorded.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- Organ weights: (see table 3)
- Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No clinical sign was recorded in the Control group. Slight laboured respiration was observed in all treatment groups in each animals during and right after the exposure. In the High dose group moderate laboured respiration was noted in few occasions in 7/10 animals. Noisy respiration was noted in all dose groups with increasing frequency and severity. Sneezing was observed in 7/10 animals of the High dose group incidentally.
Red-brown staining was also recorded in each group. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One Low dose male was found dead on Day 25 at the end of exposure. It was considered to be incidental and not related to the treatment.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control group, the body weight gain was lower in males in all dose groups (-53%, -41% and -59% in low, mid and high dose group respectively after the 4-week treatment period) with statistical significance in the Low and High dose groups and lower but without statistical significance in females (-20%, -23% and -31%).
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control group, the food consumption was lower in males in all dose groups (-16%, -13% and -20% in low, mid and high dose group respectively after the 4-week treatment period) with statistical significance in the Low and High dose groups and lower in females (-12%, -19% and -19%) with statistical significance in Mid and High dose groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no toxicologically significant effects in investigated haematology parameters between test item treated animals and the controls.
The Prothrombin Time (PTT) was significantly higher (5%) in the high dose males and mid dose females. This slight difference is not considered to be toxicologically relevant.
Occasionally, in the haematology parameters few statistical differences were seen (e.g., decreased haemoglobin in the mid dose males, decreased mean cell volume in the males of each treatment groups, decrease mean cell haemoglobin, mean platelet, absolute monocyte and absolute LUC in high dose males, increased eosinophils in mid and high dose males, increased red cell distribution width and reticulocyte relative percentage in high dose females). All groups were within the expected historical control range hence these differences were considered to be incidental. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In both sexes statistically significant differences were seen in a number of parameters, but most were without a clear dose-response or were in the normal range. There were no differences that were clearly related to treatment.
- 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):
- When compared to the controls, the following changes were observed in the mean organ weights:
The absolute and relative (to body and brain) lung weights were statistically significantly higher (p<0.01) in all male groups (by 73%, 86% and 98% in Low, Mid and High groups respectively, relative to body wt.) and in females (46%, 70% and 98% in Low, Mid and High groups respectively, relative to body wt.).
The absolute and relative (to body and brain) weights of the thymus was lower in both sexes; this is common with reduced body weight and stress.
Other organ weight statistical differences are considered to be secondary to body weight differences, or unrelated with treatment. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At necropsy, no test item-related macroscopic changes were detected at the scheduled necropsy in the Low and Mid dose groups. In the High dose group, pale diffuse discoloration in all lobes of lungs in 2/10 animals and enlargement of the lungs in 3/10 animals were noted.
In the found dead animal no test item-related macroscopic changes were detected. The red liquid material on the perinasal fur, the dark/red discoloration of the noncollapsed lungs and mandibular lymph nodes were considered as agonal. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were observed in the lungs from all dose groups. Minimal to moderate, acute alveolar/interstitial inflammation was present in 9/10 Low dose, 10/10 Mid dose and 10/10 High dose animals, with higher severity in the Mid and High dose groups. In the Low dose group the severity was minimal.
Minimal to slight, multifocal bronchiolo-alveolar hyperplasia was seen in 2/10 Low dose, 6/10 Mid dose and 10/10 High dose rats, with higher severity in the Mid and High dose groups. In the Low dose group the severity was minimal. There were no effects observed in the upper airways. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 0.04 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.04 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The exposure to the test item Supragil WP in the form of dust to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at target dose levels 0.04 to 0.2 mg/L caused laboured respiration and noisy respiration, with decreased body weight gain and food intake, plus increased lung weight at all dose levels. Moreover, acute alveolar/interstitial inflammation and multifocal bronchioloalveolar hyperplasia were observed microscopically in all dose groups. The incidence and/or severity of these histopathology changes showed dose dependency.
Based on the results of the 28d DRF study, the low dose was the proposed high dose in the subsequent 90d GLP study, but in the absence of data about the NOAEL it was necessary to repeat the 28d DRF at lower dose levels. - Executive summary:
The objective of this 4-week dose range finding study is to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item, Supragil WP, when administered to Wistar rats via inhalation route for 4 weeks with the aim of inducing toxic effects but no death or suffering at the highest concentration, and a NOAEL at the lowest concentration level. The results of the study would be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.
Five male and 5 female Wistar rats Crl:WI (Han) in each group were exposed, by a 6 hour nose-only exposure for consecutive 5 days/week, to the following target aerosol concentrations of the test item: 0.04, 0.09 and 0.2 mg/L. A control group of 5 male and 5 female rats was exposed to filtered air. . The duration of the study was 4 weeks, each week consisted of 5 consecutive treatment days and 2 days without treatment. The total number of exposures for each animal was 20. The animals were sacrificed on the day following the last exposure in Week 4. The dose levels were set based on the results of a preliminary 5-day study.
Based on the available data, the toxicity appeared to be significantly higher in males, therefore the dose levels were set to the sensitivity of the males, but they
also provided information about the dose level setting of the females for the 90-day repeated dose study.
The animals were exposed to the test atmosphere in the form milled powder using a nose-only exposure system. The test item was generated using Palas RBG1000 (Palas GmbH, Karlsruhe, Germany). Analytical concentrations of 0.043 mg/L, 0.093 mg/L and 0.216 mg/L were achieved in the respective groups.The target concentrations were 0.04 mg/L, 0.09 mg/L and 0.2 mg/L, as the Low, Mid and High Dose, respectively. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group.
Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry). Gross macroscopic examination was performed at necropsy and selected organs were weighed.
Full histopathology of the whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) was performed in each group.
The test atmosphere concentration was monitored based on the gravimetric and chemical analysis. The results of the test atmosphere characterization were considered suitable for the study purposes.
Results
One Low dose male was found dead on Day 25 at the end of exposure. It was considered to be incidental and not related to the treatment.
No clinical sign was recorded in the Control group. Laboured respiration (slight to moderate) was observed in all treatment groups during and right after the exposure, noisy respiration was noted in all dose groups with increasing frequency and severity and sneezing was observed in the High dose group.
The body weight gain was significantly lower in the males in all dose groups and it was lower but without statistical significance in the females.
The food consumption in both sexes in all dose groups was decreased compared to the control group.
In the haematology and clinical chemistry, no parameters showed effects clearly related to treatment.
At necropsy, no test item-related macroscopic changes were observed in the Low and Mid dose groups. In the High dose group, pale diffuse discoloration in all lobes of lungs in 2/10 animals and enlargement of the lungs in 3/10 animals were noted.
Lung weights were much higher than control in all dose groups.
At histology, test item-related findings were observed in the lungs from all dose groups. Minimal to moderate, acute alveolar/interstitial inflammation was present in 9/10 Low dose, 10/10 Mid dose and 10/10 High dose animals. In the Low dose group the severity was minimal, with increasing severity at the Mid and High levels.
Minimal to slight, multifocal bronchiolo-alveolar hyperplasia was seen in 2/10 Low dose, 6/10 Mid dose and 10/10 High dose rats. In the Low dose group the severity was minimal, with higher severity in the Mid and High dose groups.
Conclusion:
The exposure to the test item Supragil WP in the form of dust to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at target dose levels 0.04 to 0.2 mg/L caused laboured respiration and noisy respiration, with decreased body weight gain and food intake, plus increased lung weight at all dose levels. Moreover, acute alveolar/interstitial inflammation and multifocal bronchioloalveolar hyperplasia were observed microscopically in all dose groups. The incidence and/or severity of these histopathology changes showed dose dependency.
Based on the results of the 28d DRF study, the low dose was the proposed high dose in the subsequent 90d GLP study, but in the absence of data about the NOAEL it was necessary to repeat the 28d DRF at lower dose levels.
Referenceopen allclose all
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were regularly confirmed by using anHPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis and the nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) are presented in the following table.
Mean achieved actual and nominal test atmosphere concentrations
Gr. No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration (mg/L) |
MMAD/GSD (micron)
|
MMAD/GSD (micron) analytical |
|
Gravimetry |
HPLC analysis |
||||||
Mean of all samples |
Mean of all investigated samples |
||||||
2 |
Low |
0.004 |
0.0041 (SD: 0.0001) |
0.0042 (SD: 0.0003) |
0.0051 (SD:0.0003) |
0.61/2.91 |
0.68/2.03 |
3 |
Mid |
0.01 |
0.0104 (SD: 0.0004) |
0.0108 (SD: 0.0005) |
0.0128 (SD:0.0009) |
0.96/2.30 |
0.99/2.08 |
4 |
High |
0.04 |
0.0394 (SD: 0.0018) |
0.0393 (SD: 0.0015) |
0.0489 (SD:0.0024) |
1.16/2.09 |
1.16/1.89 |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 2.4% (max. of 7.3%), 3.8% (max. of 13.5%) and 4.6% (max. of 26.6 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.
Results of the analytical measurements were slightly higher than gravimetry by approximately 2.4% and 3.8% at the low and mid dose levels and slightly lower by 0.25% at the high dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.61-1.16 µm with Geometric Standard Deviation of 2.09-2.91.
Based on this data the test atmosphere in each treatment group is considered respirable.
Particle size distribution data of the aerosol fraction (MMAD and GSD):
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.61 |
2.91 |
94.7 |
3 |
0.96 |
2.30 |
91.5 |
4 |
1.16 |
2.09 |
90.2 |
Exposure conditions
The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres. Temperature of the test atmospheres and air were within the range during the study. The relative humidity was occasionally out of the optimal range of 30-70% or unmeasurable due to the use of water formulation for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study.
The oxygen and carbon dioxide concentrations were considered to be satisfactory for this type of study.
In conclusion, the results of the atmosphere characterization exhibited that the test atmospheres were suitable for the purposes of the study.
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were confirmed by an analytical method in the first and last week of the treatment using an HPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis are presented in the Table 5. The nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) is also presented in the following table.
Table 5: Mean achieved actual and nominal test atmosphere concentrations
Group No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration (mg/L) |
MMAD/GSD (micron) |
|
Gravimetry |
HPLC analysis |
|||||
Mean of all samples |
Mean of all investigated samples |
|||||
2 |
Low |
0.004 |
0.0041 (SD: 0.0004) |
0.0040 (SD: 0.00) |
0.0054 (SD:0.0007) |
0.63/2.62 |
3 |
Mid |
0.01 |
0.0108 (SD: 0.0013) |
0.0126 (SD: 0.00) |
0.0124 (SD:0.0011) |
1.05/2.34 |
4 |
High |
0.04 |
0.0399 (SD: 0.0026) |
0.0378 (SD: 0.01) |
0.0582 (SD:0.0093) |
1.44/1.82 |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 3.7% (max. of 15.61%), 5.9% (max. of 21.1%) and 3.0% (max. of 13.2 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.
Results of the analytical measurements were slightly higher than gravimetry by approximately 16.7% at the mid dose level and slightly lower by 5.3% at the high
dose level and 2.4% at the low dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.63-1.44 μm with Geometric Standard Deviation of 1.82-2.62. Based on this data the test atmosphere in each treatment group is considered respirable.
Table 6: Particle size distribution data of the aerosol fraction (MMAD and GSD)
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.63 |
2.62 |
94.4 |
3 |
1.05 |
2.34 |
89.8 |
4 |
1.44 |
1.82 |
88.7 |
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were confirmed byan analytical methodin the first and last week of the treatment using anHPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis are presented in the Table 5. The nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) is also presented in the following table.
Table 5: Mean achieved actual and nominal test atmosphere concentrations
Group No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration |
||
Gravimetry |
HPLC analysis |
|||||
Mean of all samples |
Mean of daily exposures |
Mean of all investigated samples |
||||
2 |
Low |
0.04 |
0.041 (SD: 0.003) |
0.041 (SD: 0.001) |
0.043 (SD: 0.04) |
0.06 (SD: 0.01) |
3 |
Mid |
0.09 |
0.091 (SD: 0.007) |
0.091 (SD: 0.003) |
0.093 (SD: 0.1) |
0.14 (SD: 0.02) |
4 |
High |
0.2 |
0.205 (SD: 0.015) |
0.205 (SD: 0.007) |
0.216 (SD: 0.58) |
0.28 (SD: 0.03) |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 2.0% (max. of 5.2%), 2.0% (max. of 8.3%) and 2.7% (max. of 9.5 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.Results of the analytical measurements were slightly higher than gravimetry by approximately 5.0% at the low, 2.2% at the mid dose levels and 5.4% at the high dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.91-1.19 µm with Geometric Standard Deviation of 2.87-3.49.
Based on this data the test atmosphere in each treatment group is considered respirable.
Table 6: Particle size distribution data of the aerosol fraction (MMAD and GSD)
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.91 |
3.16 |
90.1 |
3 |
1.19 |
3.49 |
83.0 |
4 |
1.08 |
2.87 |
90.0 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 4 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and OECD compliant studies (Klimisch 1)
- System:
- other: Body weight
Repeated dose toxicity: inhalation - local effects
Link to relevant study records
- Endpoint:
- sub-chronic toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 25 June 2017 to 23 April 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks old at the initiation of treatment
- Weight at study initiation: Males: 221-250 g; females: 156-173 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 (males) - 24 (females) days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 27.9°C
- Humidity (%): 30 - 78 %
- Air changes (per hr): 15-20 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 25 June to 30 September 2017 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.68 - <= 1.16 µm
- Geometric standard deviation (GSD):
- 2.08
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.68 (GSD = 2.03)
Group 3 (Mid dose): MMAD = 0.99 (GSD = 2.08)
Group 2 (High dose): MMAD = 1.16 (GSD = 1.89) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber was used. The rate of formulation use was controlled by a syringe pump. Verification of the target concentration, particle size and particle distribution was measured gravimetrically. Technical trials included filter analysis. Technical trials with the aqueous formulations had shown that the droplets become a powder in the dry air, so the rats were exposed principally to a powder (samples collected at the animal breathing zone were used for atmosphere characterisation).
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 13 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.004 mg/L air (nominal)
- Dose / conc.:
- 0.01 mg/L air (nominal)
- Dose / conc.:
- 0.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The dose levels were selected based on previous data available, including the results of preliminary dose range finding studies in the rat
(see 4-week studies 15/352-212PE and 15/352-212PER dated 2017-218 reported in section 7.5.2).
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. As a minimumon exposure days, individual, clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observation was performed at least twice (as
soon as practicable after removal from restraint, and approximately one hour after completion of the exposure). In non-treatment days, clinical observation was made once.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena. The first detailed observation was made on Day 8 then once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, and twice a week thereafter (except Week 1) including Day 90 (last exposure day) and fasted on Day 91.
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was conducted in all animals before treatment and on Week 13.
Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL ciclopentolato hydrochloride; Batch No.: 160515, exp.: May 2021; Batch No.: 170120, exp.: January 2022) into the conjunctival sac. The evaluation was performed by external examination and using a Gowllands or Heine Omega 500 ophthalmoscope.
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
Towards the end of the treatment period on Day 77-78, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment, on one of the 2 days per week when there was no exposure.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were also painted and measured, but they were not evaluated.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively
rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed
to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day.
The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from each dose and control group were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.
IMMUNOLOGY: No - Sacrifice and pathology:
- Gross necropsy was performed on each animal. Terminally on Day 91 surviving animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- ORGAN WEIGHTS: (see table 3)
EXAMINATION OF VAGINAL SMEARS:
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution.
The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues. - Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chisquared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate. - Clinical signs:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment related clinical signs were observed in any group.
Alopecia or fur thin were commonly recorded in all groups, transient, sporadic diuresis was recorded in males on Days 19, 20 and 23 and in females on 12, 13 and 16 in the High dose group. Sporadic appearance of scars was also observed in Low and High Dose male groups. These findings were not considered to be adverse effects of treatment. - Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control groups, there were significantly lower body weights at the end of the treatment in the Mid and High doses (9.4% and 13.1% (p<0.01) in Mid and High dose males and 5.2% (p<0.05) in High dose females) and the overall body weight gains were significantly lower in both sexes (27.3% and 37.9% in Mid and High dose males; 16.0% and 19.5% in Mid and High dose females).
It is considered that the reduced weight gain in Mid and High dose males was an adverse effect of treatment. Similarly, but to a lesser extent, High dose females were also affected; Mid dose female weight differences were similar to the High dose females, but were generally not statistically different from controls. - Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Food intakes reflected the body weight gain differences, with lower food intake in the High dose males most weeks and some weeks in the Mid dose males, with the overall
food intake significantly lower than control only in High dose males. There were no statistical differences in females. - Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. All examined animals were found to be normal.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the haematology parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is considered that there were
no adverse haematology changes in the study.
The WBC count for some control males was higher than typically seen; apparently statistically lower WBC values in the treated groups was not considered to be a
treatment effect, taking into account control data from a contemporaneous study. This difference consequentially caused the absolute WBC sub-populations to be statistically
different as well, but since the relative counts were clearly not affected by treatment, these differences were not of biological significance. There were no effects on female
WBC parameters.
Small differences in RBC, Hct, MCV or MCH were all close to the expected range without any consistent indication of a test item effect on erythrocyte parameters, these
were not considered to reflect an adverse effect of treatment. Similarly, the differences in APTT or PTT were relatively small and were similarly considered as sporadic
statistical differences and not an adverse effect of treatment.
None of the statistical differences in haematological parameters are considered to represent an adverse effect of the test item. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In the clinical chemistry parameters, there were a few statistical differences. To establish their relationship with treatment, control data from a contemporaneous study and the data from the clinical chemistry of the preliminary study with this test item were examined. The statistical differences for Creatin were clearly without a dose response.
The statistical differences for Sodium, ALT and ALKP were close to the expected control range, did not show indications of a dose response and showed no differences in the preliminary 28-day study (Report 15/353-212PE) hence these statistical differences were not considered to represent a treatment related effect. The lower Glucose and Triglycerides in High dose males, and the apparent reductions in Protein Albumin and A/G ratio in High dose females were all changes that mirrored the changes seen in the 28-day preliminary study, hence although the magnitude of the changes were not great, they were ascribed to being an effect of treatment. In the absence of any histological changes in the liver or kidney, the toxicological significance of these changes is equivocal; the magnitude of the differences suggests these are not indicative of significant adverse systemic toxicity. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity in any dose group.
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- effects observed, treatment-related
- Description (incidence and severity):
- The following treatment related changes were observed in the mean organ weights:
The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females. The lung weight relative to body weight was about 21% and 55% above control in Mid and High dose males respectively; in female Mid and High dose groups the differences were similar at about 24% and 58% respectively.
In the thymus (an organ generally sensitive to stress) there appeared to be a degree of individual stress related with treatment. The absolute and relative (to body and/or brain) thymus weights were statistically significantly lower in the Mid and High dose groups of males (abs. weights: 29.7% and 35.2% (p<0.01); rel. to bodyweights: 22.8% and 25.6% (p<0.01) and rel. to brain: 26.8% and 32.9% (p<0.01)) and in the High dose group of the females (abs. weight: 25.5% (p<0.05); rel. to brain: 26.8% (p<0.05)). Histopathology confirmed decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
Other statistical differences in organ weight were generally directly associated with the body weight differences, with lower absolute weights or higher relative weights of some organs, but with no relationship with systemic effects on the organs. The female adrenal (absolute and relative) appeared higher than control, but in the absence of any histopathological change this difference was not considered to be an adverse effect of treatment. - Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Test item-related macroscopic changes were not seen during the necropsy.
Other findings, like thin fur, scares on the skin, enlarged mediastinal lymph node (in 1/10 High dose male), dark red discoloration of the thymus (in 1/10 Low dose male) and the dilatation of uterus could be considered as background or procedure related. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium were microscopically observed in the lungs from the Mid and High dose
male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). There were no effects observed in the Low dose. The histological changes are correlated with organ weight changes. This histological change is considered to reflect a local irritation response caused by the presence of the test item in the atmosphere; the effect is considered to be an adverse effect. There were no other changes suggestive of a degenerative pathology.
Histopathology of the thymus showed decrease of size/cellularity of the thymic cortex in High dose males. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
The decreased cellularity in the thymus and other sporadic changes, like increased cellularity in the mediastinal lymph node, inflammatory cell infiltrate in the prostate,congestion/haemorrhage in the thymus, erosion/ulceration in the skin and signs of oestrus were considered as incidental or background. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Description (incidence and severity):
- Examination of vaginal smeares: There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 0.004 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- food consumption and compound intake
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.01 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused decreased
body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item.
There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m3). - Executive summary:
The objective of this 90-day study was to evaluate the potential toxic effects of the test item, Supragil WP, when administered to Wistar (Han) rats via inhalation route for at least 90 days. This study was carried out according to OECD test guideline No. 413 (September 2009), in compliance with the Good Laboratory Practices (GLP).
Three groups of 10 male and 10 female Wistar rats Crl:WI (Han) were exposed to the test item, at target concentrations of 0.004, 0.01 and 0.04 mg/L using a nose-only
exposure system. A vehicle control group of 10 male and 10 female rats was exposed to distilled water. The animals were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). Start of the exposure for all 4 groups was Day 1.
The treatment was performed in 4 inhalation towers in parallel, one tower for each treatment groups and an additional tower for the vehicle control group.
Surviving animals including controls were terminated on the day following the last exposure on Day 91.
The achieved concentrations and particle size distributions for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.
Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.
Results:
There was no mortality in the study.
No treatment related clinical signs were observed in the treatment groups.
There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity; hence no neurological effects were identified in any dose group.
There were significantly lower body weights at the end of the treatment in the Mid and High dose groups and the body weight gains were significantly lower in both sexes, particularly in males. Food intakes reflected the body weight gain differences.
No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination.
In the haematology and clinical chemistry parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is
considered that there were no adverse clinical pathology changes in the study.
There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the
oestrus phases.
The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females.
These changes correlated with a local irritation response of hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium observed microscopically in the lungs from the Mid and High dose male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). These changes were considered to be adverse effects of the test item.
An apparent stress effect was seen in thymus weights on some Mid and High dose males correlated, at histology, with a decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
There were no other changes in organ weights, necropsy or histological observations.
In summary, Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused
decreased body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item. There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m3).
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 28 December 2016 to 01 February 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8 weeks old at the initiation of treatment
- Weight at study initiation: Males: 250-282 g; females: 172-191 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 19 days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.1 – 23.5°C
- Humidity (%): 24 - 61 %
- Air changes (per hr): At least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 28 December 2016 To: 23 January 2017 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.63 - <= 1.44 µm
- Geometric standard deviation (GSD):
- 2.62
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.63 (GSD = 2.62)
Group 3 (Mid dose): MMAD = 1.05 (GSD = 2.34)
Group 2 (High dose): MMAD = 1.44 (GSD = 1.82) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber was used. The rate of formulation use was controlled by a syringe pump. Verification of the target concentration, particle size and particle distribution was measured gravimetrically. Technical trials included filter analysis. Technical trials with the aqueous formulations had shown that the droplets become a powder in the dry air, so the rats were exposed principally to a powder (samples
collected at the animal breathing zone were used for atmosphere characterisation).
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Air change rate:
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 4 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.004 mg/L air (nominal)
- Dose / conc.:
- 0.01 mg/L air (nominal)
- Dose / conc.:
- 0.04 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The concentration levels were determined based on the available experimental data from a preliminary 5 days escalating dose study and a previous 28-day DRF study (study 15/352-212PE dated 2017 reported in section 7.5.2).
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after
removal from restraint, and approximately one hour after completion of the exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal were recorded with precision of 1 g on Day 1 (before the exposure), once a week thereafter and on Days 26 and 27 (prior to necropsy, fasted weight).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All animals were euthanized under pentobarbital anaesthesia on Day 27 (RELEASE, Lot No.: 106075; Expiry: July 2018; WDT, Produced by Garbsen, Germany) by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was
observed macroscopically. All abnormalities were recorded with details of the location, colour, shape and size, as appropriate.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- Organ weights: (see table 3)
- Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No clinical sign was recorded in the Control group. Slight laboured respiration was observed in all treatment groups in some treatment days in each animal during and right after the exposure. The frequency of this observation was increased with the dose. In one High dose male decreased activity (slight) was recorded on Day 3.
Red-brown staining, wet fur, alopecia or scar were also recorded. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. - Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- There were no statistically significant differences in body weight in any group. The overall body weight gain was not statistically different to controls; although the mean values were 48% and 38% below control in the High dose males and females respectively, and at the Mid dose about 25% lower in both sexes.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food intakes reflected the body weight gain differences.
- Food efficiency:
- not examined
- 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 toxicologically significant effects in investigated haematology parameters between test item treated animals and the controls.
Occasionally, in the haematology parameters few statistical differences were seen (e.g., decreased haemoglobin, mean cell haemoglobin in the High dose males, decreased white blood cell, mean cell haemoglobin, platelet count and lymphocytes in Mid dose females). All groups were within the expected historical control range and there were no coherent trends, these statistical differences were considered to be incidental.
There was no significant change in the blood clotting parameters. - Clinical biochemistry findings:
- no effects observed
- Description (incidence and severity):
- In both sexes statistically significant differences were seen in electrolytes, protein or albumin, but most were without a clear dose-response or were in the normal range. There were no differences that were clearly related to treatment.
- 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):
- When compared to the controls, the following changes were observed in the mean organ weights:
The absolute and relative (to body and brain) lung weights were statistically significantly higher (p<0.01) in High dose group of both sexes, the relative lung weight to body weight was also higher in the male Mid dose group (p<0.05). - Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related findings were identified in the lungs from the male High dose group only. Minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats. These changes were considered to be treatment related and are in line with the lung organ weight differences observed.
Other changes, like multifocal, mononuclear cell infiltrate in the liver of 1/10 Mid dose male and signs of oestrus in 1/5 Mid and 2/5 High dose females were considered as incidental or background. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- NOAEC
- Effect level:
- 0.004 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- clinical signs
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.01 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The exposure to the test item Supragil WP in distilled water to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels 0.004 to 0.04 mg/L caused transient slight laboured respiration and noisy respiration.
There were no statistically significant effects on body weight or clinical pathology. Lung weights were significantly increased in both sexes at the High dose, with slightly higher relative weights in Mid dose males. Histologically, minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
These changes in the High Dose were similar to those observed in the Low Dose of the previous DRF (15/352-212PE).
The NOAEL is considered to be the Low dose (0.004 mg/L) although the changes in the Mid dose (0.01 mg/L) were minimal with no histopathological effects observed. Dose levels above the High dose (0.04 mg/L) are considered to be too high for a 90 day study.
Based on the results of the two 28d DRF studies, the dose levels for the subsequent 90d GLP study were set at 0.04, 0.01 and 0.004 mg/L. - Executive summary:
The objective of this additional 4-week dose range finding study is to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item when administered to Wistar rats via inhalation route for 4 weeks with the aim of inducing toxic effects but no death or suffering at the highest
concentration, and a NOAEL at the lowest concentration level. In a previous 28-day dose range finding (DRF) study (No. 15/352-212PE, Ref. 8) the NOAEL was not identified. The results of the study would be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.
Five male and 5 female Wistar rats Crl:WI (Han) in each group were exposed, by a 6 hour nose-only exposure for consecutive 5 days/week, to the following target
aerosol concentrations of the test item: 0.004, 0.01 and 0.04 mg/L. A control group of 5 male and 5 female rats was exposed to filtered air. The duration of the study was 4 weeks, each week consisted of 5 consecutive treatment days and 2 days without treatment. The total number of exposures for each animal was 20. The animals were sacrificed on the day following the last exposure in Week 4.
In the previous 28-day DRF study where 0.2 mg/L, 0.09 mg/L and 0.04 mg/L were tested, significant toxicity was observed in each dose groups and none of these
concentrations was low enough to determine the NOAEL. Therefore, in the current study, the low dose (0.04 mg/L) of the previous study was selected as high dose, and two lower concentrations were tested to find the non-toxic level.
The animals were exposed to the test atmosphere with formulations made in distilled water using a nose-only exposure system. It was not technically feasible to produce the low concentrations using the milled batch as a powder. The test item was generated using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) from 0.048 % (w/w), 0.12 % (w/w) and 0.48 % (w/w) formulations for the Low, Mid and High Doses, respectively. Analytically measured concentrations of 0.004 mg/L, 0.0126 mg/L and 0.0378 mg/L were achieved in the respective groups, the target concentrations being 0.004 mg/L, 0.01 mg/L and 0.04 mg/L. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group. Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry). Gross macroscopic examination was performed at necropsy and selected organs were weighed.
Full histopathology of the whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) was performed in each group.
The test atmosphere concentration was monitored based on the gravimetric and chemical analysis. The results of the test atmosphere characterization were
considered suitable for the study purposes.
Results
No mortality was observed in the study.
Slight transient laboured respiration was observed in all dose groups during or after the treatment, more frequently in the higher doses.
The body weight gain in the High Dose in both sexes was apparently lower than control, but without statistical significance. The same trend was seen at the Mid dose. There were no treatment related changes in clinical pathology. When compared to the controls, the following changes were observed in the mean
organ weights: Lung weights were statistically significantly higher (p<0.01) for absolute and relative weights in the High dose group in both sexes. The relative lung to body weight in male Mid dose group were also higher (p<0.05).
Test item-related macroscopic changes were not seen during the necropsy. Minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar
epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
Conclusion:
The exposure to the test item Supragil WP in distilled water to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at dose levels 0.004 to
0.04 mg/L caused transient slight laboured respiration and noisy respiration. There were no statistically significant effects on body weight or clinical pathology.
Lung weights were significantly increased in both sexes at the High dose, with slightly higher relative weights in Mid dose males. Histologically, minimal/slight hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium was present in 9/10 High dose animals. Minimal, multifocal perivascular inflammatory cell infiltrate was also seen in 4/10 High dose rats.
These changes in the High Dose were similar to those observed in the Low Dose of the previous DRF (15/352-212PE).
The NOAEL is considered to be the Low dose (0.004 mg/L) although the changes in the Mid dose (0.01 mg/L) were minimal with no histopathological effects
observed. Dose levels above the High dose (0.04 mg/L) are considered to be too high for a 90 day study.
Based on the results of the two 28d DRF studies, the dose levels for the subsequent 90d GLP study were set at 0.04, 0.01 and 0.004 mg/L.
- Endpoint:
- short-term repeated dose toxicity: inhalation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- From 19 March 2016 to 06 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- Wistar Crl:WI (Han)
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old at the initiation of treatment
- Weight at study initiation: Males: 194-226 g; females: 155-184 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days
DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 – 24.3°C
- Humidity (%): 32 - 59 %
- Air changes (per hr): At least 15 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)
IN-LIFE DATES: From: 19 March 2016 To: 14 April 2016 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure:
- nose only
- Vehicle:
- clean air
- Mass median aerodynamic diameter (MMAD):
- >= 0.91 - <= 1.19 µm
- Geometric standard deviation (GSD):
- 3.49
- Remarks on MMAD:
- Group 2 (Low dose): MMAD = 0.91 (GSD = 3.16)
Group 3 (Mid dose): MMAD = 1.19 (GSD = 3.49)
Group 2 (High dose): MMAD = 1.08 (GSD = 2.87) - Details on inhalation exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and
constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, Palas RBG1000 (Palas GmbH, Karlsruhe, Germany) rotating brush generator were used connected to pressurized air supply. Atmosphere generation was dynamic. Fresh test atmosphere from the generation system was constantly supplied to the inner plenum (distribution chamber) of the
exposure system from where was distributed to the individual exposure ports.
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Air change rate:
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.
TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port
- Samples taken from breathing zone: yes - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)
- Duration of treatment / exposure:
- The animals were exposed to an atmosphere of the test item for a period of 4 weeks.
- Frequency of treatment:
- 6 hour per day ; 5 days/week
- Dose / conc.:
- 0.04 mg/L air (nominal)
- Dose / conc.:
- 0.09 mg/L air (nominal)
- Dose / conc.:
- 0.2 mg/L air (nominal)
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- - Dose selection rationale: The concentration levels were determined based on the available experimental data from a preliminary 5 days escalating dose study.
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable - Positive control:
- Not applicable
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. Individual clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observations were performed twice (as soon as practicable after
removal from restraint, and approximately one hour after completion of the exposure.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena once a week.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal were recorded with precision of 1 g on Day 1 (before the exposure), once a week thereafter and on Days 26 and 27 (prior to necropsy, fasted weight).
FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
WATER CONSUMPTION: No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 27
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No - Sacrifice and pathology:
- All animals were euthanised under pentobarbital anaesthesia (Euthanimal 40%; Lot No.: 1409236-06; Expiry: 09-2017; Produced by Alfasan Nederland BV, Kulpersweg 9, Woerden, Netherlands) by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. All abnormalities were recorded.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4) - Other examinations:
- Organ weights: (see table 3)
- Statistics:
- Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate. - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- No clinical sign was recorded in the Control group. Slight laboured respiration was observed in all treatment groups in each animals during and right after the exposure. In the High dose group moderate laboured respiration was noted in few occasions in 7/10 animals. Noisy respiration was noted in all dose groups with increasing frequency and severity. Sneezing was observed in 7/10 animals of the High dose group incidentally.
Red-brown staining was also recorded in each group. These observations were considered to be related to the restraint and exposure procedures and, in isolation, were considered not to be of toxicological relevance. - Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One Low dose male was found dead on Day 25 at the end of exposure. It was considered to be incidental and not related to the treatment.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control group, the body weight gain was lower in males in all dose groups (-53%, -41% and -59% in low, mid and high dose group respectively after the 4-week treatment period) with statistical significance in the Low and High dose groups and lower but without statistical significance in females (-20%, -23% and -31%).
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- When compared with the control group, the food consumption was lower in males in all dose groups (-16%, -13% and -20% in low, mid and high dose group respectively after the 4-week treatment period) with statistical significance in the Low and High dose groups and lower in females (-12%, -19% and -19%) with statistical significance in Mid and High dose groups.
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- There were no toxicologically significant effects in investigated haematology parameters between test item treated animals and the controls.
The Prothrombin Time (PTT) was significantly higher (5%) in the high dose males and mid dose females. This slight difference is not considered to be toxicologically relevant.
Occasionally, in the haematology parameters few statistical differences were seen (e.g., decreased haemoglobin in the mid dose males, decreased mean cell volume in the males of each treatment groups, decrease mean cell haemoglobin, mean platelet, absolute monocyte and absolute LUC in high dose males, increased eosinophils in mid and high dose males, increased red cell distribution width and reticulocyte relative percentage in high dose females). All groups were within the expected historical control range hence these differences were considered to be incidental. - Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- In both sexes statistically significant differences were seen in a number of parameters, but most were without a clear dose-response or were in the normal range. There were no differences that were clearly related to treatment.
- 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):
- When compared to the controls, the following changes were observed in the mean organ weights:
The absolute and relative (to body and brain) lung weights were statistically significantly higher (p<0.01) in all male groups (by 73%, 86% and 98% in Low, Mid and High groups respectively, relative to body wt.) and in females (46%, 70% and 98% in Low, Mid and High groups respectively, relative to body wt.).
The absolute and relative (to body and brain) weights of the thymus was lower in both sexes; this is common with reduced body weight and stress.
Other organ weight statistical differences are considered to be secondary to body weight differences, or unrelated with treatment. - Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- At necropsy, no test item-related macroscopic changes were detected at the scheduled necropsy in the Low and Mid dose groups. In the High dose group, pale diffuse discoloration in all lobes of lungs in 2/10 animals and enlargement of the lungs in 3/10 animals were noted.
In the found dead animal no test item-related macroscopic changes were detected. The red liquid material on the perinasal fur, the dark/red discoloration of the noncollapsed lungs and mandibular lymph nodes were considered as agonal. - Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- effects observed, treatment-related
- Description (incidence and severity):
- Test item-related microscopic findings were observed in the lungs from all dose groups. Minimal to moderate, acute alveolar/interstitial inflammation was present in 9/10 Low dose, 10/10 Mid dose and 10/10 High dose animals, with higher severity in the Mid and High dose groups. In the Low dose group the severity was minimal.
Minimal to slight, multifocal bronchiolo-alveolar hyperplasia was seen in 2/10 Low dose, 6/10 Mid dose and 10/10 High dose rats, with higher severity in the Mid and High dose groups. In the Low dose group the severity was minimal. There were no effects observed in the upper airways. - Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- not specified
- Key result
- Dose descriptor:
- LOAEC
- Effect level:
- 0.04 mg/L air (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- histopathology: non-neoplastic
- organ weights and organ / body weight ratios
- Key result
- Critical effects observed:
- yes
- Lowest effective dose / conc.:
- 0.04 mg/L air (nominal)
- System:
- respiratory system: lower respiratory tract
- Organ:
- lungs
- Treatment related:
- yes
- Dose response relationship:
- yes
- Relevant for humans:
- not specified
- Conclusions:
- The exposure to the test item Supragil WP in the form of dust to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at target dose levels 0.04 to 0.2 mg/L caused laboured respiration and noisy respiration, with decreased body weight gain and food intake, plus increased lung weight at all dose levels. Moreover, acute alveolar/interstitial inflammation and multifocal bronchioloalveolar hyperplasia were observed microscopically in all dose groups. The incidence and/or severity of these histopathology changes showed dose dependency.
Based on the results of the 28d DRF study, the low dose was the proposed high dose in the subsequent 90d GLP study, but in the absence of data about the NOAEL it was necessary to repeat the 28d DRF at lower dose levels. - Executive summary:
The objective of this 4-week dose range finding study is to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item, Supragil WP, when administered to Wistar rats via inhalation route for 4 weeks with the aim of inducing toxic effects but no death or suffering at the highest concentration, and a NOAEL at the lowest concentration level. The results of the study would be used for dose level selection for a forthcoming 90-day subchronic inhalation toxicity study.
Five male and 5 female Wistar rats Crl:WI (Han) in each group were exposed, by a 6 hour nose-only exposure for consecutive 5 days/week, to the following target aerosol concentrations of the test item: 0.04, 0.09 and 0.2 mg/L. A control group of 5 male and 5 female rats was exposed to filtered air. . The duration of the study was 4 weeks, each week consisted of 5 consecutive treatment days and 2 days without treatment. The total number of exposures for each animal was 20. The animals were sacrificed on the day following the last exposure in Week 4. The dose levels were set based on the results of a preliminary 5-day study.
Based on the available data, the toxicity appeared to be significantly higher in males, therefore the dose levels were set to the sensitivity of the males, but they
also provided information about the dose level setting of the females for the 90-day repeated dose study.
The animals were exposed to the test atmosphere in the form milled powder using a nose-only exposure system. The test item was generated using Palas RBG1000 (Palas GmbH, Karlsruhe, Germany). Analytical concentrations of 0.043 mg/L, 0.093 mg/L and 0.216 mg/L were achieved in the respective groups.The target concentrations were 0.04 mg/L, 0.09 mg/L and 0.2 mg/L, as the Low, Mid and High Dose, respectively. The treatment was performed in 4 inhalation towers parallel, one tower for each treatment groups and an additional tower for the filtered air control group.
Parameters monitored for the animals during the study included mortality, clinical observations, body weight, food consumption and clinical pathology evaluation (haematology, coagulation and clinical chemistry). Gross macroscopic examination was performed at necropsy and selected organs were weighed.
Full histopathology of the whole respiratory tract from nasal cavities to lung and associated lymphoid tissue (BALT) was performed in each group.
The test atmosphere concentration was monitored based on the gravimetric and chemical analysis. The results of the test atmosphere characterization were considered suitable for the study purposes.
Results
One Low dose male was found dead on Day 25 at the end of exposure. It was considered to be incidental and not related to the treatment.
No clinical sign was recorded in the Control group. Laboured respiration (slight to moderate) was observed in all treatment groups during and right after the exposure, noisy respiration was noted in all dose groups with increasing frequency and severity and sneezing was observed in the High dose group.
The body weight gain was significantly lower in the males in all dose groups and it was lower but without statistical significance in the females.
The food consumption in both sexes in all dose groups was decreased compared to the control group.
In the haematology and clinical chemistry, no parameters showed effects clearly related to treatment.
At necropsy, no test item-related macroscopic changes were observed in the Low and Mid dose groups. In the High dose group, pale diffuse discoloration in all lobes of lungs in 2/10 animals and enlargement of the lungs in 3/10 animals were noted.
Lung weights were much higher than control in all dose groups.
At histology, test item-related findings were observed in the lungs from all dose groups. Minimal to moderate, acute alveolar/interstitial inflammation was present in 9/10 Low dose, 10/10 Mid dose and 10/10 High dose animals. In the Low dose group the severity was minimal, with increasing severity at the Mid and High levels.
Minimal to slight, multifocal bronchiolo-alveolar hyperplasia was seen in 2/10 Low dose, 6/10 Mid dose and 10/10 High dose rats. In the Low dose group the severity was minimal, with higher severity in the Mid and High dose groups.
Conclusion:
The exposure to the test item Supragil WP in the form of dust to Hannover Wistar rats for 4 weeks for 6 hours/day on a 5 day per week basis at target dose levels 0.04 to 0.2 mg/L caused laboured respiration and noisy respiration, with decreased body weight gain and food intake, plus increased lung weight at all dose levels. Moreover, acute alveolar/interstitial inflammation and multifocal bronchioloalveolar hyperplasia were observed microscopically in all dose groups. The incidence and/or severity of these histopathology changes showed dose dependency.
Based on the results of the 28d DRF study, the low dose was the proposed high dose in the subsequent 90d GLP study, but in the absence of data about the NOAEL it was necessary to repeat the 28d DRF at lower dose levels.
Referenceopen allclose all
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were regularly confirmed by using anHPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis and the nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) are presented in the following table.
Mean achieved actual and nominal test atmosphere concentrations
Gr. No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration (mg/L) |
MMAD/GSD (micron)
|
MMAD/GSD (micron) analytical |
|
Gravimetry |
HPLC analysis |
||||||
Mean of all samples |
Mean of all investigated samples |
||||||
2 |
Low |
0.004 |
0.0041 (SD: 0.0001) |
0.0042 (SD: 0.0003) |
0.0051 (SD:0.0003) |
0.61/2.91 |
0.68/2.03 |
3 |
Mid |
0.01 |
0.0104 (SD: 0.0004) |
0.0108 (SD: 0.0005) |
0.0128 (SD:0.0009) |
0.96/2.30 |
0.99/2.08 |
4 |
High |
0.04 |
0.0394 (SD: 0.0018) |
0.0393 (SD: 0.0015) |
0.0489 (SD:0.0024) |
1.16/2.09 |
1.16/1.89 |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 2.4% (max. of 7.3%), 3.8% (max. of 13.5%) and 4.6% (max. of 26.6 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.
Results of the analytical measurements were slightly higher than gravimetry by approximately 2.4% and 3.8% at the low and mid dose levels and slightly lower by 0.25% at the high dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.61-1.16 µm with Geometric Standard Deviation of 2.09-2.91.
Based on this data the test atmosphere in each treatment group is considered respirable.
Particle size distribution data of the aerosol fraction (MMAD and GSD):
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.61 |
2.91 |
94.7 |
3 |
0.96 |
2.30 |
91.5 |
4 |
1.16 |
2.09 |
90.2 |
Exposure conditions
The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres. Temperature of the test atmospheres and air were within the range during the study. The relative humidity was occasionally out of the optimal range of 30-70% or unmeasurable due to the use of water formulation for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study.
The oxygen and carbon dioxide concentrations were considered to be satisfactory for this type of study.
In conclusion, the results of the atmosphere characterization exhibited that the test atmospheres were suitable for the purposes of the study.
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were confirmed by an analytical method in the first and last week of the treatment using an HPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis are presented in the Table 5. The nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) is also presented in the following table.
Table 5: Mean achieved actual and nominal test atmosphere concentrations
Group No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration (mg/L) |
MMAD/GSD (micron) |
|
Gravimetry |
HPLC analysis |
|||||
Mean of all samples |
Mean of all investigated samples |
|||||
2 |
Low |
0.004 |
0.0041 (SD: 0.0004) |
0.0040 (SD: 0.00) |
0.0054 (SD:0.0007) |
0.63/2.62 |
3 |
Mid |
0.01 |
0.0108 (SD: 0.0013) |
0.0126 (SD: 0.00) |
0.0124 (SD:0.0011) |
1.05/2.34 |
4 |
High |
0.04 |
0.0399 (SD: 0.0026) |
0.0378 (SD: 0.01) |
0.0582 (SD:0.0093) |
1.44/1.82 |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 3.7% (max. of 15.61%), 5.9% (max. of 21.1%) and 3.0% (max. of 13.2 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.
Results of the analytical measurements were slightly higher than gravimetry by approximately 16.7% at the mid dose level and slightly lower by 5.3% at the high
dose level and 2.4% at the low dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.63-1.44 μm with Geometric Standard Deviation of 1.82-2.62. Based on this data the test atmosphere in each treatment group is considered respirable.
Table 6: Particle size distribution data of the aerosol fraction (MMAD and GSD)
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.63 |
2.62 |
94.4 |
3 |
1.05 |
2.34 |
89.8 |
4 |
1.44 |
1.82 |
88.7 |
TEST ATMOSPHERE DATA
Actual and nominal concentrations
The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters. The achieved test atmosphere concentrations were confirmed byan analytical methodin the first and last week of the treatment using anHPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis are presented in the Table 5. The nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) is also presented in the following table.
Table 5: Mean achieved actual and nominal test atmosphere concentrations
Group No. |
Group Designation |
Target Concentration (mg/L) |
Achieved Concentration (mg/L) |
Nominal Concentration |
||
Gravimetry |
HPLC analysis |
|||||
Mean of all samples |
Mean of daily exposures |
Mean of all investigated samples |
||||
2 |
Low |
0.04 |
0.041 (SD: 0.003) |
0.041 (SD: 0.001) |
0.043 (SD: 0.04) |
0.06 (SD: 0.01) |
3 |
Mid |
0.09 |
0.091 (SD: 0.007) |
0.091 (SD: 0.003) |
0.093 (SD: 0.1) |
0.14 (SD: 0.02) |
4 |
High |
0.2 |
0.205 (SD: 0.015) |
0.205 (SD: 0.007) |
0.216 (SD: 0.58) |
0.28 (SD: 0.03) |
The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 2.0% (max. of 5.2%), 2.0% (max. of 8.3%) and 2.7% (max. of 9.5 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.Results of the analytical measurements were slightly higher than gravimetry by approximately 5.0% at the low, 2.2% at the mid dose levels and 5.4% at the high dose level.
Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.
Particle size analysis
According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.91-1.19 µm with Geometric Standard Deviation of 2.87-3.49.
Based on this data the test atmosphere in each treatment group is considered respirable.
Table 6: Particle size distribution data of the aerosol fraction (MMAD and GSD)
Group |
Mean Mass Median Aerodynamic Diameter (mm) |
Geometric Standard Deviation (GSD) |
Inhalable Fraction |
2 |
0.91 |
3.16 |
90.1 |
3 |
1.19 |
3.49 |
83.0 |
4 |
1.08 |
2.87 |
90.0 |
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed
- Dose descriptor:
- NOAEC
- 4 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- GLP and OECD compliant studies (Klimisch 1)
Repeated dose toxicity: dermal - systemic effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Repeated dose toxicity: dermal - local effects
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
One Klimisch score 1 study is available for repeated dose toxicity by oral route (OECD 422 test guideline study) and was used as a key study:
In a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test by oral route performed in compliance with the GLP and in accordance with the OECD 422 test guideline, Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda (purity of 75.4%) diluted in water was administered daily by gavage to Sprague Dawley rats (10 animals/sex/dose) at doses of 0; 30; 100 and 300 mg/kg bw/d (based on the three forms of sodium isopropylnaphtalenesulphonate (mono, di and tri) content i.e. 0; 39.8; 133 and 398 mg/kg bw/d in terms of registered substance). Males were exposed 2 weeks before pairing, during pairing and until sacrifice (at least 5 weeks in total) whereas females were exposed 2 weeks before pairing, during pairing, during gestation and during lactation until day 5 post-partum (6-7 weeks in total).
There were no unscheduled deaths in control or test item-treated groups. Emaciated appearance was observed in few males from 100 mg/kg bw/d. Hypoactivity and loud/abdominal breathing was observed in some females exposed to 300 mg/kg bw/d. Statistically significant decreases in body weight changes were observed in males from 100 mg/kg bw/d for the period of day 1 to 8, resulting in slight body weight loss at 300 mg/kg bw/d. This was associated with a statistically significant decrease in mean food consumption at 300 mg/kg bw/d for the period of days 1 to 8. In females, there was neither effect on body weight nor on food consumption. A minimal increase in adrenal weights was observed in females given the test item at 300 mg/kg bw/d. But in absence of microscopic correlates, this variation was considered to be of equivocal significance.
Based on the results of this study, 133 mg/kg bw/d of test item (as registered) was established as the no-observed-adverse effect-level (NOAEL) considering the clinical signs observed in males.
Three Klimish score 1 studies are available for repeated dose toxicity study by the inhalation route: one 90 -day study used as key study and two 4 -week studies (preliminary studies to the 90-day study) reported as supporting study (detailed in section 7.5.2).
In the key study, performed in compliance with GLP and according to OECD 413 test guideline, the registered substance was administered via inhalation route (nose-only) to Hannover Wistar rats at 0.004, 0.01 and 0.04 mg/L atmosphere concentration (10 animals/sex/dose). The animals were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks. Decreased body weight gain and food consumption was reported at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item. There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for the registered substance is considered to be 0.004 mg/L.
Justification for classification or non-classification
Harmonized classification:
No harmonized classification is available according to the Regulation (EC) No 1272/2008.
Self classification:
Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda is not classified for Specific Target Organ Toxicity - Repeated Exposure (STOT-RE) according to regulations (EC) 1272/2008 (CLP) and GHS UN. Indeed, by the oral route, adverse effects were only observed from the dose level of 398 mg/kg/day which is, according to CLP criteria,above the upper limit of the guidance value range for STOT-RE classification with a 28 -day study. By the inhalation route, the adverse effects were limited to slight effect on body weight and food consumption, which are not related to spectific target organ, and to changes in the respiratory tract indicative of local irrating effects already covered by the classification as STOT SE 3, H335.
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