2,2-dimethyl-1,3-dioxolan-4-ylmethanol

Endpoint summary

Description of key information

NOAEL (oral route) for systemic toxicity = 1000 mg/kg bw/day for males since the observed effects in males are not relevant for human risk assessment.
NOEL (oral route) for systemic toxicity = 1000 mg/kg bw/day for females based on the absence of significant effects related to test item on females.

NOAEC (inhalation route) for systemic and local effects = 5 mg/L (no relevant to human adverse effects reported)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 13 March 2013 to 19 November 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD Guideline 422 without any major deviation.

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:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Certificat n° 2012/96 ; 10 January 2013

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 99.9%
- Composition of test material, percentage of components: 99.9% 2,2-Dimethyl-1,3-dioxolane-4-methanol; water: 0.02%; Acidity (Acetic acid): 0.0021%
- Lot/batch No.: BR12K853
- Expiration date of the lot/batch: 05 November 2013
- Storage condition of test material: at room temperature

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Italia, Calco, Italy.
- Age at study initiation: Males: 10 weeks; Females: 9 weeks
- Weight at study initiation: Males: 388 g (331 to 440 g); females: 230 g (194 to 264 g)
- Housing: Individually, except during pairing and lactation, in polycarbonate cages (Tecniplast 2154, 940 cm²) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Toward the end of gestation and during lactation, autoclaved wood shavings (SICSA, Alfortville, France) were provided to females and their litter as nesting material.
- Diet: SSNIFF R/M-H pelleted maintenance diet (SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: Drinking water filtered with a 0.22 µm filter, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Humidity: 50 ± 20%
- Air changes: 12 air changes per hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From 19 March 2013 to 10 May 2013.

Route of administration:

oral: gavage

Vehicle:

physiological saline

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a solution in the vehicle. The test item was dissolved with the required quantity of vehicle to give the required concentrations of 50, 100 and 200 mg/mL. The formulations were prepared for up to 9 days, divided into daily aliquots and stored at room temperature and protected from light. For each dosing, daily aliquots were delivered to the study room protected from light

VEHICLE
- Concentration in vehicle: 50, 100 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- The concentrations of the dose formulations were checked in study weeks 1, 3 and 6 by a validated analytical method (CiToxLAB France/Study No. 39832 VAA).
- Acceptance criterion: measured concentration = nominal concentration ± 10%
- The pH of the obtained solutions used throughout the study was measured by means of a pH-meter.
- Results: The test item concentrations in the administered dose formulations analyzed in weeks 1, 3 and 6 were within an acceptable range of variation (i.e. -2% to +8%) when compared to the nominal values (± 10%); pH was close to neutrality

Duration of treatment / exposure:

- Males: 2 weeks before pairing, during the pairing period (up to 5 days), until sacrifice (5 weeks in total).
- Females: 2 weeks before pairing, during the pairing period (up to 5 days), during gestation, during lactation until day 5 post partum (p.p.) inclusive.
- Day 1 corresponds to the first day of the treatment period.

Frequency of treatment:

Once daily

Remarks:

Doses / Concentrations:
250, 500 or 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were selected in agreement with the Sponsor, following the results of a previous 2-week toxicity study performed with the same species at the dose-levels of 250, 500 or 1000 mg/kg bw/day (Study No. 39834 TSR). In this study, no toxicologically relevant findings, but a slight decrease in body weight in females given 1000 mg/kg bw/day when compared to controls was observed.
- Rationale for animal assignment: during the acclimation period, the required number of animals (40 males and 40 females) was selected according to body weight and clinical condition. The animals were allocated to groups (by sex) according to computerized stratification procedure based on body weight, so that the average body weight of each group was similar.

Positive control:

Not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS (PARENTAL ANIMALS): Yes
- Time schedule:
Viability / Mortality: Once a day before the treatment period and twice a day during the treatment period. A female of group 4 showing signs of poor clinical condition (immobilized hindlimbs), was humanely sacrificed on day 4 p.c., and was subjected to a macroscopic post-mortem examination.
Clinical Signs: Once daily, during acclimatization and up to day of necropsy. As a male from the control group displayed liquid feces on day 22, decision was taken to weigh and to examine the animal closely on day 23.

DETAILED CLINICAL OBSERVATIONS (PARENTAL ANIMALS): Yes
- Time schedule: Detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study. Observations included (but were not limited to) changes in the skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behavior (e.g. self-mutilation, walking backwards) were also recorded.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS): Yes
- Time schedule: The first five males and the first five females to be sacrificed on day 6 p.p. from each group were evaluated once at the end of the treatment period. For females, this was performed on day 5 p.p. after sacrifice of the pups.
- Animals were observed for the following:
Cage-side observations: "touch escape" or ease of removal from the cage.
Hand-held observations: fur appearance, salivation, lacrimation, piloerection, exophthalmos, reactivity to handling, pupil size (presence of myosis or mydriasis).
Standard arena (2-minute recording) observations: grooming, palpebral closure, defecation, urination, tremors, twitches, tonic and clonic convulsions, gait, arousal (hypo- and hyper- activity), posture, stereotypy, behavior, breathing, ataxia and hypotonia.
Reactivity to manipulation and to different stimuli: 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.
Motor activity: Finally, motor activity was measured once by automated infra-red sensor equipment over a 60-minute period.

OPHTHALMOSCOPIC EXAMINATION: No

BODY WEIGHT (PARENTAL ANIMALS): Yes
- Time schedule for examinations: The body weight of each male was recorded on the first day of treatment (day 1), then once a week until sacrifice. The body weight of each female was recorded on the first day of treatment (day 1), then once a week until mated, on days 0, 7, 14 and 20 post-coitum (p.c.) and days 1 and 5 p.p..
- The body weight of each animal sacrificed as scheduled after the end of the pairing period for males or on day 6 p.p. for females was recorded before sacrifice.

FOOD CONSUMPTION (PARENTAL ANIMALS): Yes
- Time schedule for examinations: 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 the pairing period, during pregnancy for the intervals days 0-7, 7-14 and 14-20 p.c. and during lactation for the interval days 1-5 p.p.

HAEMATOLOGY (PARENTAL ANIMALS): Yes
- Time schedule for collection of blood: from the first five males and the first five females to be sacrificed on day 6 p.p. from each group on the day of sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Blood samples were taken from the orbital sinus of the animals, under light isoflurane anesthesia, into appropriate tubes.
- Animals fasted: Yes; an overnight period of at least 14 hours
- How many animals: five males and five females
- Parameters checked: Erythrocytes, Mean cell volume, Packed cell volume, Hemoglobin, Mean cell hemoglobin Concentration, Mean cell hemoglobin, Thrombocytes, Leucocytes, Differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes and large unstained cells, monocytes), Reticulocytes, Prothrombin time, Fibrinogen, Activated partial thromboplastin time.

CLINICAL CHEMISTRY (PARENTAL ANIMALS): Yes
- Time schedule for collection of blood: from the first five males and the first five females to be sacrificed on day 6 p.p. from each group on the day of sacrifice.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Blood samples were taken from the orbital sinus of the animals, under light isoflurane anesthesia, into appropriate tubes.
- Animals fasted: Yes; an overnight period of at least 14 hours
- How many animals: five males and five females
- Parameters checked: Sodium, Potassium, Chloride , Calcium, Inorganic phosphorus, Glucose, Urea, Creatinine, Bilirubin (total), Cholesterol (total), Triglycerides, Aspartate aminotransferase, Alanine aminotransferase, Alkaline phosphatase, Protein (total), Albumin, Albumin/Globulin ratio and Bile acids

URINALYSIS: No

PREGNANCY AND PARTURITION:
- Females were allowed to litter normally and rear their progeny until day 5 p.p.. Any sign of a difficult or prolonged parturition was recorded. The morning when the parturition was completed was designated day 1 p.p.. The length of gestation was calculated.

Sacrifice and pathology:

SACRIFICE (PARENTAL ANIMALS)
- On completion of the treatment period, after at least 14 hours fasting, all surviving F0 animals were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination: males: after the end of the pairing period (5 weeks of treatment in total); females: on day 6 p.p..
- One female was prematurely sacrificed during the gestation period. This female was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination.

GROSS PATHOLOGY: Yes
- All parent animals were submitted to complete macroscopic post-mortem examination, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death. This included examination of the external surfaces, all orifices, the cranial
cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. Special attention was paid to the reproductive organs.

ORGAN WEIGHTS
Body weight of each animal was recorded before sacrifice and the organs specified in the table 7.5.1/2 were weighed. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

HISTOPATHOLOGY: Yes
- Samples of the tissues specified in the table 7.5.1/2 were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in modified Davidson's fixative).
- All tissues required for microscopic examination were trimmed, embedded in paraffin wax and sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). Microscopic examination was performed on all tissues listed in the table 7.5.1/2 from the first five sacrificed as scheduled males and the first five females sacrificed on day 6 p.p. of the control and high-dose groups (groups 1 and 4) and from the prematurely sacrificed female, and on all macroscopic lesions of all groups. Based on the microscopic findings seen in the kidneys of high-dose males, a microscopic examination was performed on the kidneys from group 2 and group 3 males. Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Other examinations:

No data

Statistics:

- Data are expressed as group mean values ± standard deviation (body weight, body weight change, food consumption or as proportions. Whenever appropriate, the experimental unit of comparison was the litter. Data of the non-pregnant female are not included in group mean calculations such as body weight, body weight change, food consumption.
- Body weight, food consumption and reproductive data are 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).
- Hematology and blood biochemistry data are compared by various tests according to a decision tree: Dunnett-test, Dunn test or Mann-Whitney / Wilcoxon test (according to variance homogeneity between groups) were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups. Mann-Whitney / Wilcoxon test or Dunn test were applied when the data could not be assumed to follow a normal distribution.
- PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01) by Dunn test if >2 groups or Wilcoxon test if 2 groups and Dunnett test if >2 groups or t-test if 2 groups, based on normal distribution and homogeneity of variance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
- There were no deaths considered as test item-related. One female given 1000 mg/kg/day was prematurely sacrificed on day 4 p.c. on ethical grounds as this animal displayed hindlimb immobilized before sacrifice. No necropsy findings were noted. In view of the duration of the treatment, and absence of pre-neoplastic or neoplastic hematopoietic lesions in other test item-treated animals, this isolated finding was considered to be fortuitous. There were no other unscheduled deaths during the study.
- There were no test item treatment-related clinical signs.

BODY WEIGHT AND WEIGHT GAIN (PARENTAL ANIMALS)
- There were no test item treatment-related effects on mean body weight or mean body weight gain.
- When compared with the control group, a slightly lower mean body weight gain was observed in females treated at 1000 mg/kg/day during the whole pre-mating period (+30 g and +18 g between Days 1 – 15, respectively). When compared with the control group, the opposite trend was noted in all test item-treated males with a statistical significance over the last 3 weeks at 250 and 1000 mg/kg/day (+31 g, +50 g and +49 g between Days 15 – 36, respectively). Since these differences were not dose-related and of opposite tendency and since these differences had no impact on the final body weights, they were considered not to be relevant.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- When compared with the mean control values, females treated at 500 and 1000 mg/kg/day had statistically significantly lower food consumption (23 g/rat/day, 20 g/rat/day and 21 g/rat/day between Days 1 – 8 of Pre-mating, respectively). As this effect was minimal, not dose-related and limited to the first week of the pre-mating period, it was considered to be of no toxicological importance. There were no effects on mean food consumption at these dose-levels in males.
- The test item did not affect the food consumption at any of the dose-levels employed.

NEUROBEHAVIOURAL EXAMINATION (PARENTAL ANIMALS)
- No relevant findings were noted during functional observational battery testing in test item-treated groups when compared with the control groups.

LABORATORY INVESTIGATIONS (PARENTAL ANIMALS)
- There were no test item-related findings on hematology parameters. Lower mean values of Mean Cell Hemoglobin, prothrombin and activated partial thromboplastin times recorded in males given the high dose-level were considered to be of no toxicological importance as they were minimal, with no biological significance and/or isolated variations.
- When compared with the mean control values, the cholesterol concentration of males treated at 1000 mg/kg/day was 77% higher (2.3 vs. 1.3 mmol/L, p<0.01). In absence of treatment-related microscopic findings in the liver, these variations were considered to be of minor toxicological importance.



ORGAN WEIGHTS (PARENTAL ANIMALS)
- There were no test item-related organ weight changes in females. Test item-related changes were observed for the liver in males. When compared with controls, there was a slight increase in the mean absolute and relative liver weights in males given the test item at 1000 mg/kg/day (+31% and +26%, respectively), reaching statistical significance for the relative weight only. A minimal trend was also present at 500 mg/kg/day (+22% and + 18%, respectively), but the differences were not statistically significant. These variations were considered to be related to the test item but were considered not to be adverse in view of the slight magnitude of the changes.
- The mean absolute and relative spleen weights were slightly higher in males given the test item at 1000 mg/kg/day compared with controls (up to +29%). This correlated at microscopic examination with increased congestion in two animals, and was considered to be an agonal phenomenon related to euthanasia with pentobarbital.
- Other occasional organ weight changes were not considered to be related to the test item as they were small in amplitude, had no gross or microscopic correlates, and/or were not dose-related in magnitude.

GROSS PATHOLOGY (PARENTAL ANIMALS)
- In the vagina of the female given 1000 mg/kg/day prematurely sacrificed on day 4 p.c., there was a slight subacute inflammation, and moderate mucification of the mucosal epithelium. Low development of the corpora lutea in the ovaries and slight glandular atrophy in the stomach (fundus) may have been secondary to stress and poor clinical condition of this animal. None of these findings were attributed to the test item.
- There were no macroscopic findings attributed to the test item administration. The thymus was reduced in size in a single female in each of the 500 and 1000 mg/kg/day dose-groups.
This correlated at microscopic examination with lymphoid atrophy. However lymphoid atrophy was recorded with similar incidence in controls and high-dose females. These macroscopic findings were therefore considered to be incidental and unrelated to the test item administration.

HISTOPATHOLOGY (PARENTAL ANIMALS)

- There were no test item-related findings in females. Test item-related microscopic findings occurred in the kidneys from males. In the kidneys, tubular hyaline droplets were seen with increased incidence and severity in males given the test item at 1000 mg/kg/day compared with controls. This was characterized by the presence of dense eosinophilic droplets in proximal tubular epithelium. These hyaline droplets, occasionally seen in untreated male rats, are consistent with α2u globulin. Although human excrete proteins of a similar nature, they are found in only trace amounts and therefore this finding is considered to be non-relevant for human. There were no significant findings at 250 and 500 mg/kg/day.
- Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, had no dose-relationship in incidence or severity, and/or are common background findings for the Sprague-Dawley rat.

Key result

Dose descriptor:

NOAEL

Remarks:

(Systemic toxicity)

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

None

Conclusions:

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day in rats (based on findings in the kidneys of males).

Executive summary:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in Phosphate Buffer Saline solution was administered daily by oral gavage to male and female Sprague-Dawley rats (10/sex/dose), for 2 weeks before mating, during mating and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 250, 500 or 1000 mg/kg bw/day. A control group was treated with Phosphate Buffer Saline solution. During the study, data was recorded on mortality, clinical signs, behavioural assessments, body weight change, food consumption, haematology, blood chemistry, mating performance, fertility and gestation length. All animals were subjected to a gross necropsy examination, selected organs were weighed and histopathological evaluation of selected tissues was performed.

 

No test item-related deaths or clinical signs were noted in any group and sex. No toxicologically relevant effects on mean body weight, mean food consumption, mean Functional Observation Battery and motor activity data and mean clinical pathology parameters in any group and sex. The increase in the absolute and relative liver weights recorded in males given 1000 mg/kg bw/day was considered not to be adverse in view of the slight magnitude of the changes and the absence of microscopic correlates. None of the macroscopic findings were attributed to the test item administration. At histopathology, tubular hyaline droplets in the kidneys (consistent with rat-specific alpha-2u-globulin) were seen with increased incidence and severity in males treated at 1000 mg/kg bw/day compared to controls (presence of dense eosinophilic droplets in proximal tubular epithelium). This finding was not observed at 250 or 500 mg/kg bw/day. These kidney effects were considered to be related to alpha-2u globulin nephropathy and of no relevance to humans.

 

Under the test conditions, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day in rats (based on findings in the kidneys of males).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Recent GLP study conducted according to OECD Guideline 422 without any major deviation (Klimisch score = 1).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 December 2017 to 11 October 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 20170320 provided by Solvay
- Expiration date of the lot/batch: 20 April 2018
- Purity test date: 20 March 2017
- Purity: 99.66%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

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, SandhoferWeg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: , approx. 7-8 weeks old at the initiation of treatment.
- Weight at study initiation: Males: 190 - 222 g ; Females: 141 - 175 g.
- Fasting period before study: No.
- Housing: Rodents were housed 2-3 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.
- Diet: Animals received ssniff® SM R/M "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch: 382 24962; Exp.: 04/2018), ad libitum.
- Water (e.g. ad libitum): Animals received tap water from municipal supply, as for human consumption from a 500 ml bottles, ad libitum while in their home cages.
- Acclimation period: 5 (males) - 12 (females) days

DETAILS OF FOOD AND WATER QUALITY:
- The food supplier provided analytical certificate for the batch used, which are archived with the study raw data.
- Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1–24.9°C
- Humidity (%): 32–57%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 19 December 2017 To: 26 March 2018

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 2.35 - <= 2.41 µm

Geometric standard deviation (GSD):

1.4

Remarks on MMAD:

MMAD/GSD (micron) (see details in Results section):
Low dose: 2.35/2.61
Mid dose: 2.41/1.90
High dose: 2.39/1.84

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 stainless steel cylinders, the inner plenum and the outer chamber with 10 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/relative humidity, O2 and CO2 sensors.
These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn.
- 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: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where the aerosol was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: The flow of air through each port was at least 0.5 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- Air change rate: not specified
- Method of particle size determination:
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol 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).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

From the samples and impactors collected during the animal exposure for gravimetric concentration measurement, a confirmatory chemical analysis was performed using GC-FID method validated under Study code: 16/379-316AN. The filter analysis was performed weekly during for the first 4 weeks, then every 3 weeks during the experimental period. Frequency was agreed with the Sponsor.

- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ACTUAL TEST ATMOSPHERE 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). 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). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).

The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:

CAS = M / (tS x FS)

Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

From the samples and impactors collected during the animal exposure for gravimetric concentration measurement, a confirmatory chemical analysis was performed using GC-FID method validated under Study code: 16/379-316AN. The filter analysis was performed weekly during for the first 4 weeks, then every 3 weeks during the experimental period. Frequency was agreed with the Sponsor.

NOMINAL TEST ATMOSPHERE CONCENTRATIONS:
Nominal concentrations were calculated by dividing the total weight of test item disseminated into the test chamber by the total volume of air used during the same period.

PARTICLE SIZE ANALYSIS:
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 in the first 4 weeks then in every 4th week at each concentration tested and measured gravimetrically and confirmed by validated GC-FID method (Citoxlab Hungary study code: 16/379-316AN) together with filter sample analysis. 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 respirable in the rat).
In the Low Dose group, the impactors were cooled down in freezer prior sampling to prevent evaporation during sampling.

Duration of treatment / exposure:

The animals were exposed to an atmosphere for a period of 13 weeks.

Frequency of treatment:

6 hours per day ; 5 days/week basis (6 days in the first week).

Dose / conc.:

0.5 mg/L air (nominal)

Dose / conc.:

1.5 mg/L air (nominal)

Dose / conc.:

5 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor in consultation with the Study Director, based on previous data available, including the results of a preliminary dose range finding study in the rat (Citoxlab Hungary study code 16/379-212PE) (assigned as supporting study in Iuclid section 7.5.2). In the Phase 1 of the DRF study, the animals were exposed to a test item atmosphere at the target concentration of 5.0 mg/L for 3 consecutive days (1.5, 3 and 6 hours/day). The actual concentration was 4.99 mg/L. Since no clinical signs were observed in this concentration, the Phase 2 of the DRF was performed in two target atmosphere concentrations (5.0 mg/L and 1.25 mg/L) in two inhalation systems and three male and three female Wistar Hannover rats Crl:WI(Han) were exposed to the test atmospheres in each tower for 3 and 6 hours per day on a 7 day per week basis for a period of 2 weeks (total study duration of 14 days). The mean actual concentrations were 5.22 mg/L and 1.34 mg/L. Based on the results of the DRF study, it is considered that the high dose (limit level) of 5 mg/L is suitable, and that it is likely that the low dose level of 0.5 mg/L of AUGEO SL 191 will not have significant adverse effects.
- Rationale for animal assignment (if not random): All animals were allocated to study groups based on their most recent body weights. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomisation.
- Rationale for selecting satellite groups: Not applicable

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks were made twice daily, early and late during the normal working day, for mortality and/or morbidity amongst the test animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As a minimum on 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 were made on all animals outside the home cage in a standard arena. The first detailed observation was made on Day 1 then once a week.
- Cage side observations checked included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, on Day 1 and weekly thereafter 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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Ophthalmoscopic examination was conducted in all animals before treatment and in the control and High Dose animals on Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, after an overnight period of food deprivation, prior to scheduled necropsy on Day 91
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, after an overnight period of food deprivation, prior to scheduled necropsy on Day 91
- Animals fasted: Yes
- How many animals: all animals except #4005 and #3502 euthanized at termination.
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: The evaluation of the urine samples was performed by observation (colour/appearance) or test strips as applicable. In case of animal #4502, there was not enough sample for evaluation, in case of animal #2001, there was no evaluation of sedimentation
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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.
- Dose groups that were examined: all
- Battery of functions tested: 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.

IMMUNOLOGY: No

OTHER: 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Macroscopic examination was performed for all animals (see details in table 5).
HISTOPATHOLOGY: Yes. Full histopathology was performed in Control and High dose groups. In addition, lungs were investigated in Mid and Low dose groups. Tissues and organs with macroscopic findings were also evaluated. (see details in table 5).

Other examinations:

ORGAN WEIGHT MEASUREMENT (see table 4)

IMMUNOHISTOCHEMISTRY: Alpha(2)-microglobulin expression (to determine if there is pathology mediated by alpha-2 microglobulin nephropathy) was evaluated in the kidney from control and High Dose treated male rats at the end of the treatment. Left Kidney samples (longitudinal sections) embedded in paraffin wax (paraffin blocks) were used for α-2u globulin analysis. Sections were cut and transferred to slides. Tissue sections were immunostained for α-2u globulin and examined.

Statistics:

Group mean and standard deviation were calculated for numerical data. 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 show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (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 was the better option when the normality and heterogeneity assumptions implicit in the tests are 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 were 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 Chi-squared 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 treated groups. These findings were not considered to be adverse effects of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related adverse effect in the body weight or body weight gain data. It is remarkable that when compared with the control groups, body weights in High dose females was higher than the control at each point during the study (from 1.6% - 7.5%), but these differences were not significant and the data were within the historical control range.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intakes reflected the body weight gain difference, with higher food intake in the High dose females during the study. There were no statistical differences in the food consumption data. The food consumption was not evaluated in cages when spillage was recorded.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the haematology parameters, there were a few statistical differences when compared with the Control, but not consistent between sexes or clearly related to treatment and all were within the historical control range. It is considered that there were no adverse haematology changes in the study (see summary results in table 6).

In the males, PTT values were significantly lower in Mid and High dose groups, but they were within the historical control range. In the females, haemoglobin in Mid dose, MCV and basophil (rel.) in High dose were significantly different, but all were within the historical control range (see summary results in table 6).

None of these sporadic 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 no statistical differences in the males, but a few statistical differences were found in the females.
The triglyceride values were significantly lower in all dose groups in females, but they were not dose-related and they were within the historical control range. In the Low and Mid dose females AST/GOT was significantly higher (statistically significant only in the Low dose), while ALT/GPT was significantly higher in Low and Mid doses. These values did not show dose response, they were not statistically significantly different in males and they were within the historical control range (see summary results in table 7).
As they were not consistent between sexes or clearly related to treatment and all were within the historical control range, the clinical chemistry changes were considered not to be adverse.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was statistically significantly higher (p < 0.01) urine pH of female animals in the Mid and High doses. Due to the lack of microscopic findings in the kidneys of the females, they were considered non adverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related effect in the Landing Foot Splay or Irwin Test in any dose group. Although, some significant changes were measured in the Grip Strength and locomotor activity, these changes were within the historical control range and not considered to be test item related adverse effect.

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 (see summary results in table 8):
The absolute and relative (to body and brain) liver weights were statistically significantly higher in the High dose groups both in the males and females, but without microscopic correlates. The absolute and/or relative (to body and brain) kidney weights were statistically significantly higher in the Mid and High dose females (without microscopic correlates). In males, only the relative kidney weights were statistically significantly higher at High dose (correlated with microscopic findings). The absolute and relative (to body and/or brain) adrenal weights were statistically significantly higher in the High dose group of females. In the Mid dose group of the females, only the absolute weight was statistically significantly higher than in the Control. These variations were not associated with any microscopic changes. The absolute weight of the brain increased in the High dose females as well, but without microscopic changes.

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, dilated renal pelvis, pale, or dark red discoloration of the lungs, red foci in the glandular mucosa of the stomach, dark red discoloration of the thymus 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):

The increased incidence and severity of eosinophilic droplets in the renal cortex of the High dose males (1/10 minimal, 9/10 slight) compared to Controls (3/10 minimal, 3/10 slight) are considered as test item-related. Immuno-staining confirmed that the histological changes seen in High dose male kidneys were related to alpha-2 microglobulin nephropathy, as such the change is not relevant for human exposure. Since the change is not adverse it was not considered necessary to examine the Mid or Low dose male kidney slides histologically.

Other changes, like tubular basophilia (3/10 High dose males) and multifocal, inflammatory cell infiltrate (1/10 High dose male) in the kidneys, congestion/hemorrhage in the thymus, liver and gastric mucosa, the centrilobular hepatocellular hypertrophy, acute inflammation in the lungs (1/10 High dose male), inflammatory cell infiltrate in the prostate, tubular atrophy/degeneration in the testis and signs of estrus are considered as incidental or background.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

IHC evaluation of α-2u globulin in kidney hyaline droplets:
When compared with controls, there was an increased severity of α-2u globulin-positive hyaline droplets in the tubular epithelium of High dose males. This finding is indicative of alpha-2 microglobulin nephropathy, a phenomenon exclusively found in adult male rats

Key result

Dose descriptor:

NOAEC

Effect level:

> 5 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Highest dose tested (limit dose)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

 

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 an GC-FID 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	GC-FID analysis
			Mean of all samples	Mean of all investigated samples
2	Low	0.5	0.55 (SD: 0.03)	0.46 (SD: 0.05)	1.43 (SD:0.13)	2.35/2.61	2.20/2.61
3	Mid	1.5	1.53 (SD: 0.06)	1.49 (SD: 0.17)	5.74 (SD:0.28)	2.41/1.90	2.30/1.85
4	High	5.0	5.08 (SD: 0.14)	4.98 (SD: 0.37)	23.3 (SD:1.13)	2.39/1.84	2.42/1.80

 

The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 5.5%, 3.9% and 2.8% 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 lower than gravimetry by approximately 16.4%, 2.6% and 2.0 at the low, mid and high dose levels, respectively.

 

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 2.35-2.41 µm with Geometric Standard Deviation of 1.84-2.61.

The calculation of the last two gravimetric particle size analysis in the Low dose group were not valid, as the last stages of the impactors did not contain detectable amount of Test Item, but the analytical determinations from the very same impactors were successful and the particle size distributions could be calculated based on that.

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 (MMAD) (mm)	Geometric Standard Deviation (GSD)	Inhalable Fraction (% < 3mm)
2	2.35	2.61	60.1
3	2.41	1.90	63.7
4	2.39	1.84	64.5

 

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 acceptable range during the study, except in few occasions in the Control group (min. of 18.7^oC). Due to the properties of the Test Item, the relative humidity was occasionally lower than the optimal range of 30-70%. 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.

Table 6: Summary of hematology results

	Atmosphere target concentration (mg/L)				Historical Control Data
	Control	0.5	1.5	5.0	Mean    Min-Max
Males
Haemoglobin (g/dL)	15.50	15.30	15.33	15.22	15.68     14.7-16.8
	differences %	-1.3	-1.1	-1.8
MCV (fL)	54.31	55.08	54.74	54.32	53.43     50.5-58.7
	differences %	1.4	0.8	0.0
Basophils Rel. (%)	0.21	0.12	0.17	0.42	0.14       0.0-1.0
	differences %	-42.9	-19.0	100.0
Prothrombin time (PTT, second)	10.21	10.08	9.95^dd	9.80^dd	10.20     9.4-11.0
	differences %	-1.3	-2.5	-4.0
Females
Haemoglobin (g/dL)	15.29	15.10	14.70^d	15.06	15.15    14.0-16.5
	differences %	-1.2	-3.9	-1.5
MCV (fL)	56.16	56.27	57.26	57.79^u	54.95    51.8-59.1
	differences %	0.2	2.0	2.9
Basophils Rel. (%)	0.72	0.51	0.76	0.22^u	0.20       0.0-1.0
	differences %	-29.2	5.6	-69.4
Prothrombin time (PTT, second)	9.80	9.62	9.62	9.59	9.53       8.9-10.3
	differences %	-1.8	-1.8	-2.1

^u= Dunn-Test 2 sided p < 0.05

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

 

Table 7: Summary of clinical chemistry results

	Atmosphere target concentration (mg/L)				Historical Control Data
	Control	0.5	1.5	5.0	mean    min-max
Males
TRIG (mmol/L)	0.499	0.438	0.488	0.512	0.452      0.16-0.98
	differences %	-12.2	-2.2	2.6
AST/GOT (U/L)	168.9	202.3	209.1	342.4	146.0      88-332
	differences %	19.8	23.8	102.7
ALT/GPT (U/L)	72.8	94.2	99.2	204.8	65.8        38-169
	differences %	29.4	36.3	181.3
Females
TRIG (mmol/L)	0.316	0.252^dd	0.258^d	0.262^d	0.332      0.10-1.09
	differences %	-20.3	-18.4	-17.1
AST/GOT (U/L)	150.4	263.1^u	270.9	180.7	169.9      101-658
	differences %	74.9	80.1	20.1
ALT/GPT (U/L)	45.1	95.8^uu	100.6^u	71.2	55.3        32-193
	differences %	112.4	123.0	57.9

^u= Dunn-Test 2 sided p < 0.05

^uu= Dunn-Test 2 sided p < 0.01

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

Table 8: Summary of body weight variations

		Atmosphere concentration (mg/L)
	Control	0.5	1.5	5.0
Males
Liver (g)	7.511	7.606	8.165	8.708^d
	differences (%)	1.3	8.7	15.9
Liver/BW (%)	2.298	2.250	2.391	2.674^uu
	differences (%)	-2.1	4.1	16.4
Liver/Brain (%)	363.23	366.74	393.55	417.67^d
	differences (%)	1.0	8.3	15.5
Kidneys (g)	1.893	2.059	2.048	2.180
	differences (%)	8.8	8.2	15.2
Kidneys/BW (%)	0.584	0.611	0.600	0.671^u
	differences (%)	4.7	2.8	14.9
Kidneys/Brain (%)	91.73	99.40	98.75	105.16
	differences (%)	8.4	7.6	14.6
Adrenals (g)	0.0603	0.0617	0.0670	0.0655
	differences (%)	2.3	11.1	8.6
Adrenals/BW (%)	0.0186	0.0183	0.0196	0.0202
	differences (%)	-1.4	5.7	8.8
Adrenals/Brain (%)	2.920	2.979	3.225	3.162
	differences (%)	2.0	10.4	8.3
Brain (g)	2.066	2.070	2.077	2.074
	differences (%)	0.2	0.5	0.4
Females
Liver (g)	5.081	5.594	5.560	5.962^dd
	differences (%)	10.1	9.4	17.3
Liver/BW (%)	2.656	2.838	2.829	2.940^dd
	differences (%)	6.9	6.5	10.7
Liver/Brain (%)	263.13	286.96	280.98	295.89^d
	differences (%)	9.1	6.8	12.4
Kidneys (g)	1.286	1.352	1.395^u	1.467^uu
	differences (%)	5.1	8.5	14.1
Kidneys/BW (%)	0.674	0.687	0.711	0.723^d
	differences (%)	1.9	5.5	7.3
Kidneys/Brain (%)	66.62	69.28	70.53	72.75^dd
	differences (%)	4.0	5.9	9.2
Adrenals (g)	0.0675	0.0775	0.0785^u	0.0836^uu
	differences (%)	14.8	16.3	23.9
Adrenals/BW (%)	0.0354	0.0392	0.0400	0.0411^d
	differences (%)	10.5	13.0	16.1
Adrenals/Brain (%)	3.499	3.975	3.974	4.148^uu
	differences (%)	13.6	13.6	18.5
Brain (g)	1.931	1.952	1.979	2.016^dd
	differences (%)	1.1	2.5	4.34

^u= Dunn-Test 2 sided p < 0.05

^uu= Dunn-Test 2 sided p < 0.01

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

Conclusions:

AUGEO SL 191 administered via inhalation route to Hannover Wistar for 90 days at 0.5, 1.5 and 5.0 mg/L atmosphere concentration, caused increased incidence and severity of the eosinophilic droplets in the renal cortex of the High dose males. This finding was considered to be non-adverse, but a well-known male rat specific change (alpha-2 microglobulin nephropathy).
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.

In conclusion, under the conditions of this sub-chronic inhalation toxicity study, the No Observed Adverse Effect Concentration (NOAEC) for AUGEO SL 191 is considered to be 5.0 mg/L (limit value).

Executive summary:

The objective of this 90-day study was to evaluate the potential toxic effects of the test item, AUGEO SL 191, 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 0.5, 1.5 and 5.0 mg/L using a nose-only exposure system. A control group of 10 male and 10 female rats was exposed to clean air. The animalswere 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 air 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. The Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 2.35-2.41 µm with Geometric Standard Deviation of 1.84-2.61. Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.

There was no mortality in the study. No treatment related clinical signs were observed in the treatment groups. There was no treatment related effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity; hence no neurological effects were identified in any dose group. There was no treatment related adverse effect in the body weight, body weight gain and food consumption data. No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. In the haematology, clinical chemistry and urinalysis 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. There were statistically significant absolute and/or relative (to body and brain) liver, kidney, brain and adrenal weight changes, but as they were without histopathological correlates, they were considered non adverse. In the High dose males, increased incidence and severity of eosinophilic droplets in the kidneys were observed by histopathology. The immunohistochemical investigation showed that these droplets contain alpha-2 microglobulin and are indicative of alpha-2 microglobulin nephropathy, a phenomenon exclusively found in adult male rats.

 

In conclusion, under the conditions of this sub-chronic inhalation toxicity study, the No Observed Adverse Effect Concentration (NOAEC) for AUGEO SL 191 is considered to be 5.0 mg/L (limit value).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 30 October 2017 to 23 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

14-Day Dose Range Finding study to 90-Day inhalation study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

shorter duration (14-day instead of 28-day)

GLP compliance:

no

Remarks:

Dose Range Finding study performed to determine dose levels of a subsequent 90-day study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 20170320 provided by Solvay
- Expiration date of the lot/batch: 20 March 2018
- Purity test date: 20 March 2017
- Purity: 99.66%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 7 weeks old at the initiation of Phase 1 and 8 weeks old at the initiation of Phase 2.
- Weight at study initiation: Phase 1: Males: 194-214 g; females: 158-163 g / Phase 2: Males: 221-246 g; females: 160-180 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet (e.g. ad libitum): 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 ad libitum.
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: at least five days prior to involvement in the study

DETAILS OF FOOD AND WATER QUALITY: see above

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 24.5°C
- Humidity (%): 31 - 58 %
- 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: 30 October 2017 To: 27 November 2017

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: untreated

Mass median aerodynamic diameter (MMAD):

>= 2.61 - <= 2.87 µm

Geometric standard deviation (GSD):

1.83

Remarks on MMAD:

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L (v.s. target concentration of 5.0 mg/L);, the MMAD was 2.87 micron (GSD: 1.94).
In the Phase 2, the mean achieved atmospheric concentrations were 1.34 and 5.22 mg/L (v.s. target concentrations of 1.25 and 5.0 mg/L, respectively); the MMAD were 2.66 micron (GSD: 1.95) and 2.61 micron (GSD: 1.83), for the low and high dose, respectively.

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. Two identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric stainless steel cylinders, the inner plenum and the outer chamber with 10 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.
- 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).
- Method of conditioning air: not specified
- System of generating particulates/aerosols: 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: Test atmosphere temperature, Test atmosphere relative humidity, Test atmosphere carbon dioxide concentration and Test atmosphere oxygen concentration
were monitored during each exposure period by the TSE-DACO monitoring system. The test atmosphere temperature, relative humidity, oxygen and carbon dioxide concentration were considered to be satisfactory for this type of study.
- Air flow rate: The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- Air change rate: not specified
- 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: see section "Details on analytical verification of doses or concentrations"
- Samples taken from breathing zone: yes (see above)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on the preliminary data, the non-volatile content of the test item is nearly 0 after 24 hours, but the volatility is quite slow in short period, therefore 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). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
Where
M = Mass of the active substance on the sorbent (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)
In addition, the actual concentration will bewas verified by a validated GC method (16/379-316AN). Samples of the test atmosphere were collected through a G10 filter, as described above, or similar) and analysed once in Phase 2, for each dose group. Samples were collected from a vacant animal exposure port (animals breathing zone) and at least 3 samples at each concentration level were analysed at each occasion. Samples were analysed on the day of the sampling

Duration of treatment / exposure:

In Phase 1, single male and female Wistar rats Crl:WI (Han) were exposed nose-only to fixed target aerosol concentration of 5.0 mg/L for 1.5 hour, 3 hour and 6 hours per day.
In Phase 2, the animals were exposed to an atmosphere of the test item for 3 or 6 hours per day.

Frequency of treatment:

In Phase 1, animals were exposed for 3 Days.
In Phase 2, animals were exposed for 14 Days.

Dose / conc.:

4.99 mg/L air (analytical)

Remarks:

Phase 1 animals

Dose / conc.:

1.34 mg/L air (analytical)

Remarks:

Phase 2 animals

Dose / conc.:

5.22 mg/L air (analytical)

Remarks:

Phase 2 animals

No. of animals per sex per dose:

Phase 1: 1 animal/sex/exposure duration
Phase 2: 3 animals/sex/dose/exposure duration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The starting dose level was set based on acute toxicity data where no mortality was reported up to the highest dose of 5 mg/L air.
- Rationale for animal assignment: randomisation based on body weights
- 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:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were performed prior to exposure and once during 1.5 and 3 hour exposures and twice during the 6 hour exposure whilst the animals were still restrained. 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).
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: In Phase 1, body weights were measured daily. In Phase 2, the body weight of each animal was recorded at randomization, then on Day 1 (before the exposure), and twice a week thereafter and on Day 15 (prior to necropsy, fasted).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes : Food consumption was recorded in Phase 2 weekly and the weekly food consumption was calculated in Phase 2.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY/BLOOD CLOTTING: Yes
- Time schedule for collection of blood: Only in Phase 2: Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia (on Day 15)
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Not specified
- How many animals: all phase 2 animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No. 2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: Gross necropsy was performed on each animal. Terminally (Day 4 in Phase 1 and Day 15 in Phase 2), surviving animals were euthanised under pentobarbital anaesthesia by exsanguination.
In the Phase 1 animals only macroscopic observation was performed.
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.
In the Phase 2, the macroscopic examination of the tissues and organs listed in Table no. 3 were retained from all animals.

HISTOPATHOLOGY: Full histopathology was not performed.

Other examinations:

Organ weight measurements:
The following organs were weighed in all Phase 2 animals: Brain, Lungs, Adrenals, Epididymis, Spleen, Ovaries,Heart, Testes, Kidneys, Liver, Thymus, Uterus including cervix.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups is checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett’s (Multiple Range) test is used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests show significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis is required. A Kruskal-Wallis analysis of variance is used after Rank Transformation. If there is a positive result, the inter-group comparisons are performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in the body weight of males at the end of the treatment in the Low dose, Low dose 2 and High dose groups, but without dose response. The body weight gain of males was also significantly lower in the treatment groups comparing to the control, also without dose response. These differences are considered non-treatment related, but related to the restraint procedure, as the control animals were non restrained, while the treated animals spent 3 or 6 hours in restraint tube, resulting in different shape of their body weight curve

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Lower food consumption was reported in test item treated groups compared with the control. This change reflected the body weight gain differences between treated and control groups comparing to the control and are considered non-treatment related, but related to the restraint procedure.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to the controls, the following changes were observed in the mean organ weights:
The relative to brain liver weight in the males of the High Dose group (p<0.05) and in each female group was statistically significantly lower (p<0.01 in Low dose, p<0.05 in the other groups). In females, adrenal gland weight relative to bodyweight in Low dose and High dose groups (p<0.05), and the brain weight relative to bodyweight in Low dose, High dose and High dose 2 groups were statistically significantly (p<0.05) higher.
These changes were without any dose response, therefore they are considered to be incidental and non treatment related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

> 5 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported up to the highest dose tested

Critical effects observed:

no

Test Atmosphere data

Actual and nominal concentrations

The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters.

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L, the MMAD was 2.87 micron (GSD: 1.94).

The achieved test atmosphere concentrations were confirmed byan analytical methodusing a GCmethod in Phase 2. The mean achieved actual test atmosphere concentrations in Phase 2 based on the gravimetry and specific analysis are presented in the table below. 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.

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	GC analysis
			Mean of all samples	Mean of all investigated samples
2	Low Dose 1 and 2	1.25	1.3442 (SD: 0.0739)	1.1828 (SD:0.07)	5.3089 (SD:0.3978)	2.66/1.95
4	High Dose 1 and 2	5.0	5.2204 (SD: 0.1552)	4.7959 (SD: 0.17)	25.8878 (SD:1.2243)	2.61/1.83

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 5.5% and 2.97% in the Low 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 lower than gravimetry by approximately 12.0% at the Low dose level and 8.13% 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.

The particle size distribution data werewithin the range[DN1] in both phases of the study.

Based on these data the test atmosphere in each treatment group is considered respirable.

Exposure conditions

The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres.

The temperature, humidity, oxygen and carbon dioxide values 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.

Conclusions:

The exposure to the test item AUGEO SL 191 to Hannover Wistar rats for 2 weeks for 3 and 6 hours/day on a 7 day per week basis at dose levels 1.25 mg/L to 5.0 mg/L did not cause any clinical sign or any treatment related effect in the body weight, clinical pathology or histopathology. The Maximum Tolerated Dose (MTD) and the No Observed Adverse Effect Level (NOAEL) are considered to be higher than the High dose (5.0 mg/L) in this study.

Executive summary:

The objective of this 14-day study was to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for 14 days with the aim of inducing toxic effects but no death or suffering at the highest concentration, and a NOAEC at the lowest concentration level. The results of the study are for dose level selection for a subsequent 90-day inhalation toxicity study.

 

In Phase 1, single male and female Wistar rats Crl:WI (Han) were exposed nose-only to fixed target aerosol concentration of 5.0 mg/L for 1.5 hour, 3 hours and 6 hours in the first day. As no clinical signs were observed, the exposure was continued at this concentration for two more consecutive days. The objective of this phase was to find the maximum tolerated concentration (MTC) and to determine suitable concentrations for Phase 2.

Based on the results of Phase 1, two atmosphere concentrations, 1.25 and 5.0 mg/L, were set up in two inhalation systems and three male and three female Wistar rats Crl:WI (Han) were exposed to the test atmosphere in each tower for 3 and 6 hours per day on a 7 day per week basis for a period of 2 weeks (total study duration of 14 days).

A control group of three male and three females was an untreated group.

Surviving animals including controls were terminated on the day following the last exposure on Day 15.

The animals were exposed to the test atmosphere with liquid aerosol using the Test Item as supplied. The test item was generated using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany).

 

In Phase 1, mortality, clinical observations and body weight was measured.

 

In Phase 2, 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 in Phase 1 and Phase 2 and chemical analysis in Phase 2. The results of the test atmosphere characterization were considered suitable for the study purposes.

Results

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L (v.s. target concentration of 5.0 mg/L); the MMAD was 2.87 micron (GSD: 1.94).

In the Phase 2, the mean achieved atmospheric concentrations were 1.34 and 5.22 mg/L (v.s. target concentrations of 1.25 and 5.0 mg/L, respectively); the MMAD were 2.66 micron (GSD: 1.95) and 2.61 micron (GSD: 1.83), for the low and high dose, respectively.

No mortality and no clinical signs observed in the study.

The body weight gain was lower in each treatment groups comparing to the control, but without dose response. It was due to the restraint and treatment procedure, as the control animals were not treated on inhalation tower.

Lower food consumption was reported in test item treated groups compared with the control. This change reflected the body weight gain differencesbetween treated and control groups and was considered non-treatment related. .

There were no treatment related changes in clinical pathology.

There was no treatment related effect in the organ weights.

There were no treatment related macroscopic observation and no effect on the respiratory tract.

 

In conclusion, the exposure to the test item AUGEO SL 191 to Hannover Wistar rats for 2 weeks for 3 and 6 hours/day on a 7 day per week basis at dose levels 1.25 mg/L to 5.0 mg/L did not cause any clinical signs or any treatment related effect in the body weight, clinical pathology or histopathology.

The MTD and the NOAEL are considered to be higher than the High dose (5.0 mg/L) in this study. As 5.0 mg/L is the maximum limit concentration in inhalation studies with liquid aerosols, no testing at higher dose levels is required.

Based on the results of the DRF study, the dose levels for the subsequent 90d GLP study should be 5.0, 1.5 and 0.5 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

5 000 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and OECD 413 compliant study.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 19 December 2017 to 11 October 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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 20170320 provided by Solvay
- Expiration date of the lot/batch: 20 April 2018
- Purity test date: 20 March 2017
- Purity: 99.66%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

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, SandhoferWeg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: , approx. 7-8 weeks old at the initiation of treatment.
- Weight at study initiation: Males: 190 - 222 g ; Females: 141 - 175 g.
- Fasting period before study: No.
- Housing: Rodents were housed 2-3 animals of the same sex and group/cage. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities.
- Diet: Animals received ssniff® SM R/M "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany (Batch: 382 24962; Exp.: 04/2018), ad libitum.
- Water (e.g. ad libitum): Animals received tap water from municipal supply, as for human consumption from a 500 ml bottles, ad libitum while in their home cages.
- Acclimation period: 5 (males) - 12 (females) days

DETAILS OF FOOD AND WATER QUALITY:
- The food supplier provided analytical certificate for the batch used, which are archived with the study raw data.
- Water quality control analysis was performed at least once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).
The food and water were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.1–24.9°C
- Humidity (%): 32–57%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 19 December 2017 To: 26 March 2018

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 2.35 - <= 2.41 µm

Geometric standard deviation (GSD):

1.4

Remarks on MMAD:

MMAD/GSD (micron) (see details in Results section):
Low dose: 2.35/2.61
Mid dose: 2.41/1.90
High dose: 2.39/1.84

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 stainless steel cylinders, the inner plenum and the outer chamber with 10 circularly arranged exposure ports.
The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/relative humidity, O2 and CO2 sensors.
These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn.
- 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: not specified
- Method of conditioning air: not specified
- System of generating particulates/aerosols: Atmosphere generation was dynamic. Fresh aerosol from the generation system was constantly supplied to the inner plenum (distribution chamber) of the exposure system from where the aerosol was distributed to the individual exposure ports. After passing through the animal’s breathing zone, spent aerosol entered the outer cylinder from where it was exhausted through a suitable filter system.
- Temperature, humidity, pressure in air chamber:
- Air flow rate: The flow of air through each port was at least 0.5 L/min. This flow rate was considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- Air change rate: not specified
- Method of particle size determination:
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol 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).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

From the samples and impactors collected during the animal exposure for gravimetric concentration measurement, a confirmatory chemical analysis was performed using GC-FID method validated under Study code: 16/379-316AN. The filter analysis was performed weekly during for the first 4 weeks, then every 3 weeks during the experimental period. Frequency was agreed with the Sponsor.

- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ACTUAL TEST ATMOSPHERE 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). 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). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).

The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:

CAS = M / (tS x FS)

Where
M = Mass of the active substance on the filter (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)

From the samples and impactors collected during the animal exposure for gravimetric concentration measurement, a confirmatory chemical analysis was performed using GC-FID method validated under Study code: 16/379-316AN. The filter analysis was performed weekly during for the first 4 weeks, then every 3 weeks during the experimental period. Frequency was agreed with the Sponsor.

NOMINAL TEST ATMOSPHERE CONCENTRATIONS:
Nominal concentrations were calculated by dividing the total weight of test item disseminated into the test chamber by the total volume of air used during the same period.

PARTICLE SIZE ANALYSIS:
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 in the first 4 weeks then in every 4th week at each concentration tested and measured gravimetrically and confirmed by validated GC-FID method (Citoxlab Hungary study code: 16/379-316AN) together with filter sample analysis. 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 respirable in the rat).
In the Low Dose group, the impactors were cooled down in freezer prior sampling to prevent evaporation during sampling.

Duration of treatment / exposure:

The animals were exposed to an atmosphere for a period of 13 weeks.

Frequency of treatment:

6 hours per day ; 5 days/week basis (6 days in the first week).

Dose / conc.:

0.5 mg/L air (nominal)

Dose / conc.:

1.5 mg/L air (nominal)

Dose / conc.:

5 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Dose selection rationale:
The dose levels were selected by the Sponsor in consultation with the Study Director, based on previous data available, including the results of a preliminary dose range finding study in the rat (Citoxlab Hungary study code 16/379-212PE) (assigned as supporting study in Iuclid section 7.5.2). In the Phase 1 of the DRF study, the animals were exposed to a test item atmosphere at the target concentration of 5.0 mg/L for 3 consecutive days (1.5, 3 and 6 hours/day). The actual concentration was 4.99 mg/L. Since no clinical signs were observed in this concentration, the Phase 2 of the DRF was performed in two target atmosphere concentrations (5.0 mg/L and 1.25 mg/L) in two inhalation systems and three male and three female Wistar Hannover rats Crl:WI(Han) were exposed to the test atmospheres in each tower for 3 and 6 hours per day on a 7 day per week basis for a period of 2 weeks (total study duration of 14 days). The mean actual concentrations were 5.22 mg/L and 1.34 mg/L. Based on the results of the DRF study, it is considered that the high dose (limit level) of 5 mg/L is suitable, and that it is likely that the low dose level of 0.5 mg/L of AUGEO SL 191 will not have significant adverse effects.
- Rationale for animal assignment (if not random): All animals were allocated to study groups based on their most recent body weights. There was an equal number of animals from each weight group in each of the experimental groups assigned by randomisation.
- Rationale for selecting satellite groups: Not applicable

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks were made twice daily, early and late during the normal working day, for mortality and/or morbidity amongst the test animals.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: As a minimum on 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 were made on all animals outside the home cage in a standard arena. The first detailed observation was made on Day 1 then once a week.
- Cage side observations checked included changes in the skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, on Day 1 and weekly thereafter 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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups that were examined: Ophthalmoscopic examination was conducted in all animals before treatment and in the control and High Dose animals on Week 13.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, after an overnight period of food deprivation, prior to scheduled necropsy on Day 91
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, after an overnight period of food deprivation, prior to scheduled necropsy on Day 91
- Animals fasted: Yes
- How many animals: all animals except #4005 and #3502 euthanized at termination.
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: The evaluation of the urine samples was performed by observation (colour/appearance) or test strips as applicable. In case of animal #4502, there was not enough sample for evaluation, in case of animal #2001, there was no evaluation of sedimentation
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: 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.
- Dose groups that were examined: all
- Battery of functions tested: 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.

IMMUNOLOGY: No

OTHER: 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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Macroscopic examination was performed for all animals (see details in table 5).
HISTOPATHOLOGY: Yes. Full histopathology was performed in Control and High dose groups. In addition, lungs were investigated in Mid and Low dose groups. Tissues and organs with macroscopic findings were also evaluated. (see details in table 5).

Other examinations:

ORGAN WEIGHT MEASUREMENT (see table 4)

IMMUNOHISTOCHEMISTRY: Alpha(2)-microglobulin expression (to determine if there is pathology mediated by alpha-2 microglobulin nephropathy) was evaluated in the kidney from control and High Dose treated male rats at the end of the treatment. Left Kidney samples (longitudinal sections) embedded in paraffin wax (paraffin blocks) were used for α-2u globulin analysis. Sections were cut and transferred to slides. Tissue sections were immunostained for α-2u globulin and examined.

Statistics:

Group mean and standard deviation were calculated for numerical data. 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 show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunnett’s (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 was the better option when the normality and heterogeneity assumptions implicit in the tests are 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 were 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 Chi-squared 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 treated groups. These findings were not considered to be adverse effects of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related adverse effect in the body weight or body weight gain data. It is remarkable that when compared with the control groups, body weights in High dose females was higher than the control at each point during the study (from 1.6% - 7.5%), but these differences were not significant and the data were within the historical control range.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Food intakes reflected the body weight gain difference, with higher food intake in the High dose females during the study. There were no statistical differences in the food consumption data. The food consumption was not evaluated in cages when spillage was recorded.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the haematology parameters, there were a few statistical differences when compared with the Control, but not consistent between sexes or clearly related to treatment and all were within the historical control range. It is considered that there were no adverse haematology changes in the study (see summary results in table 6).

In the males, PTT values were significantly lower in Mid and High dose groups, but they were within the historical control range. In the females, haemoglobin in Mid dose, MCV and basophil (rel.) in High dose were significantly different, but all were within the historical control range (see summary results in table 6).

None of these sporadic 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 no statistical differences in the males, but a few statistical differences were found in the females.
The triglyceride values were significantly lower in all dose groups in females, but they were not dose-related and they were within the historical control range. In the Low and Mid dose females AST/GOT was significantly higher (statistically significant only in the Low dose), while ALT/GPT was significantly higher in Low and Mid doses. These values did not show dose response, they were not statistically significantly different in males and they were within the historical control range (see summary results in table 7).
As they were not consistent between sexes or clearly related to treatment and all were within the historical control range, the clinical chemistry changes were considered not to be adverse.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was statistically significantly higher (p < 0.01) urine pH of female animals in the Mid and High doses. Due to the lack of microscopic findings in the kidneys of the females, they were considered non adverse.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

There was no treatment related effect in the Landing Foot Splay or Irwin Test in any dose group. Although, some significant changes were measured in the Grip Strength and locomotor activity, these changes were within the historical control range and not considered to be test item related adverse effect.

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 (see summary results in table 8):
The absolute and relative (to body and brain) liver weights were statistically significantly higher in the High dose groups both in the males and females, but without microscopic correlates. The absolute and/or relative (to body and brain) kidney weights were statistically significantly higher in the Mid and High dose females (without microscopic correlates). In males, only the relative kidney weights were statistically significantly higher at High dose (correlated with microscopic findings). The absolute and relative (to body and/or brain) adrenal weights were statistically significantly higher in the High dose group of females. In the Mid dose group of the females, only the absolute weight was statistically significantly higher than in the Control. These variations were not associated with any microscopic changes. The absolute weight of the brain increased in the High dose females as well, but without microscopic changes.

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, dilated renal pelvis, pale, or dark red discoloration of the lungs, red foci in the glandular mucosa of the stomach, dark red discoloration of the thymus 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):

The increased incidence and severity of eosinophilic droplets in the renal cortex of the High dose males (1/10 minimal, 9/10 slight) compared to Controls (3/10 minimal, 3/10 slight) are considered as test item-related. Immuno-staining confirmed that the histological changes seen in High dose male kidneys were related to alpha-2 microglobulin nephropathy, as such the change is not relevant for human exposure. Since the change is not adverse it was not considered necessary to examine the Mid or Low dose male kidney slides histologically.

Other changes, like tubular basophilia (3/10 High dose males) and multifocal, inflammatory cell infiltrate (1/10 High dose male) in the kidneys, congestion/hemorrhage in the thymus, liver and gastric mucosa, the centrilobular hepatocellular hypertrophy, acute inflammation in the lungs (1/10 High dose male), inflammatory cell infiltrate in the prostate, tubular atrophy/degeneration in the testis and signs of estrus are considered as incidental or background.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

IHC evaluation of α-2u globulin in kidney hyaline droplets:
When compared with controls, there was an increased severity of α-2u globulin-positive hyaline droplets in the tubular epithelium of High dose males. This finding is indicative of alpha-2 microglobulin nephropathy, a phenomenon exclusively found in adult male rats

Key result

Dose descriptor:

NOAEC

Effect level:

> 5 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: Highest dose tested (limit dose)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

5 mg/L air (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

 

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 an GC-FID 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	GC-FID analysis
			Mean of all samples	Mean of all investigated samples
2	Low	0.5	0.55 (SD: 0.03)	0.46 (SD: 0.05)	1.43 (SD:0.13)	2.35/2.61	2.20/2.61
3	Mid	1.5	1.53 (SD: 0.06)	1.49 (SD: 0.17)	5.74 (SD:0.28)	2.41/1.90	2.30/1.85
4	High	5.0	5.08 (SD: 0.14)	4.98 (SD: 0.37)	23.3 (SD:1.13)	2.39/1.84	2.42/1.80

 

The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 5.5%, 3.9% and 2.8% 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 lower than gravimetry by approximately 16.4%, 2.6% and 2.0 at the low, mid and high dose levels, respectively.

 

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 2.35-2.41 µm with Geometric Standard Deviation of 1.84-2.61.

The calculation of the last two gravimetric particle size analysis in the Low dose group were not valid, as the last stages of the impactors did not contain detectable amount of Test Item, but the analytical determinations from the very same impactors were successful and the particle size distributions could be calculated based on that.

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 (MMAD) (mm)	Geometric Standard Deviation (GSD)	Inhalable Fraction (% < 3mm)
2	2.35	2.61	60.1
3	2.41	1.90	63.7
4	2.39	1.84	64.5

 

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 acceptable range during the study, except in few occasions in the Control group (min. of 18.7^oC). Due to the properties of the Test Item, the relative humidity was occasionally lower than the optimal range of 30-70%. 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.

Table 6: Summary of hematology results

	Atmosphere target concentration (mg/L)				Historical Control Data
	Control	0.5	1.5	5.0	Mean    Min-Max
Males
Haemoglobin (g/dL)	15.50	15.30	15.33	15.22	15.68     14.7-16.8
	differences %	-1.3	-1.1	-1.8
MCV (fL)	54.31	55.08	54.74	54.32	53.43     50.5-58.7
	differences %	1.4	0.8	0.0
Basophils Rel. (%)	0.21	0.12	0.17	0.42	0.14       0.0-1.0
	differences %	-42.9	-19.0	100.0
Prothrombin time (PTT, second)	10.21	10.08	9.95^dd	9.80^dd	10.20     9.4-11.0
	differences %	-1.3	-2.5	-4.0
Females
Haemoglobin (g/dL)	15.29	15.10	14.70^d	15.06	15.15    14.0-16.5
	differences %	-1.2	-3.9	-1.5
MCV (fL)	56.16	56.27	57.26	57.79^u	54.95    51.8-59.1
	differences %	0.2	2.0	2.9
Basophils Rel. (%)	0.72	0.51	0.76	0.22^u	0.20       0.0-1.0
	differences %	-29.2	5.6	-69.4
Prothrombin time (PTT, second)	9.80	9.62	9.62	9.59	9.53       8.9-10.3
	differences %	-1.8	-1.8	-2.1

^u= Dunn-Test 2 sided p < 0.05

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

 

Table 7: Summary of clinical chemistry results

	Atmosphere target concentration (mg/L)				Historical Control Data
	Control	0.5	1.5	5.0	mean    min-max
Males
TRIG (mmol/L)	0.499	0.438	0.488	0.512	0.452      0.16-0.98
	differences %	-12.2	-2.2	2.6
AST/GOT (U/L)	168.9	202.3	209.1	342.4	146.0      88-332
	differences %	19.8	23.8	102.7
ALT/GPT (U/L)	72.8	94.2	99.2	204.8	65.8        38-169
	differences %	29.4	36.3	181.3
Females
TRIG (mmol/L)	0.316	0.252^dd	0.258^d	0.262^d	0.332      0.10-1.09
	differences %	-20.3	-18.4	-17.1
AST/GOT (U/L)	150.4	263.1^u	270.9	180.7	169.9      101-658
	differences %	74.9	80.1	20.1
ALT/GPT (U/L)	45.1	95.8^uu	100.6^u	71.2	55.3        32-193
	differences %	112.4	123.0	57.9

^u= Dunn-Test 2 sided p < 0.05

^uu= Dunn-Test 2 sided p < 0.01

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

Table 8: Summary of body weight variations

		Atmosphere concentration (mg/L)
	Control	0.5	1.5	5.0
Males
Liver (g)	7.511	7.606	8.165	8.708^d
	differences (%)	1.3	8.7	15.9
Liver/BW (%)	2.298	2.250	2.391	2.674^uu
	differences (%)	-2.1	4.1	16.4
Liver/Brain (%)	363.23	366.74	393.55	417.67^d
	differences (%)	1.0	8.3	15.5
Kidneys (g)	1.893	2.059	2.048	2.180
	differences (%)	8.8	8.2	15.2
Kidneys/BW (%)	0.584	0.611	0.600	0.671^u
	differences (%)	4.7	2.8	14.9
Kidneys/Brain (%)	91.73	99.40	98.75	105.16
	differences (%)	8.4	7.6	14.6
Adrenals (g)	0.0603	0.0617	0.0670	0.0655
	differences (%)	2.3	11.1	8.6
Adrenals/BW (%)	0.0186	0.0183	0.0196	0.0202
	differences (%)	-1.4	5.7	8.8
Adrenals/Brain (%)	2.920	2.979	3.225	3.162
	differences (%)	2.0	10.4	8.3
Brain (g)	2.066	2.070	2.077	2.074
	differences (%)	0.2	0.5	0.4
Females
Liver (g)	5.081	5.594	5.560	5.962^dd
	differences (%)	10.1	9.4	17.3
Liver/BW (%)	2.656	2.838	2.829	2.940^dd
	differences (%)	6.9	6.5	10.7
Liver/Brain (%)	263.13	286.96	280.98	295.89^d
	differences (%)	9.1	6.8	12.4
Kidneys (g)	1.286	1.352	1.395^u	1.467^uu
	differences (%)	5.1	8.5	14.1
Kidneys/BW (%)	0.674	0.687	0.711	0.723^d
	differences (%)	1.9	5.5	7.3
Kidneys/Brain (%)	66.62	69.28	70.53	72.75^dd
	differences (%)	4.0	5.9	9.2
Adrenals (g)	0.0675	0.0775	0.0785^u	0.0836^uu
	differences (%)	14.8	16.3	23.9
Adrenals/BW (%)	0.0354	0.0392	0.0400	0.0411^d
	differences (%)	10.5	13.0	16.1
Adrenals/Brain (%)	3.499	3.975	3.974	4.148^uu
	differences (%)	13.6	13.6	18.5
Brain (g)	1.931	1.952	1.979	2.016^dd
	differences (%)	1.1	2.5	4.34

^u= Dunn-Test 2 sided p < 0.05

^uu= Dunn-Test 2 sided p < 0.01

^d= Dunnett 2 Sided p < 0.05

^dd= Dunnett 2 Sided p < 0.01

Conclusions:

AUGEO SL 191 administered via inhalation route to Hannover Wistar for 90 days at 0.5, 1.5 and 5.0 mg/L atmosphere concentration, caused increased incidence and severity of the eosinophilic droplets in the renal cortex of the High dose males. This finding was considered to be non-adverse, but a well-known male rat specific change (alpha-2 microglobulin nephropathy).
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.

In conclusion, under the conditions of this sub-chronic inhalation toxicity study, the No Observed Adverse Effect Concentration (NOAEC) for AUGEO SL 191 is considered to be 5.0 mg/L (limit value).

Executive summary:

The objective of this 90-day study was to evaluate the potential toxic effects of the test item, AUGEO SL 191, 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 0.5, 1.5 and 5.0 mg/L using a nose-only exposure system. A control group of 10 male and 10 female rats was exposed to clean air. The animalswere 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 air 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. The Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 2.35-2.41 µm with Geometric Standard Deviation of 1.84-2.61. Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.

There was no mortality in the study. No treatment related clinical signs were observed in the treatment groups. There was no treatment related effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity; hence no neurological effects were identified in any dose group. There was no treatment related adverse effect in the body weight, body weight gain and food consumption data. No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. In the haematology, clinical chemistry and urinalysis 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. There were statistically significant absolute and/or relative (to body and brain) liver, kidney, brain and adrenal weight changes, but as they were without histopathological correlates, they were considered non adverse. In the High dose males, increased incidence and severity of eosinophilic droplets in the kidneys were observed by histopathology. The immunohistochemical investigation showed that these droplets contain alpha-2 microglobulin and are indicative of alpha-2 microglobulin nephropathy, a phenomenon exclusively found in adult male rats.

 

In conclusion, under the conditions of this sub-chronic inhalation toxicity study, the No Observed Adverse Effect Concentration (NOAEC) for AUGEO SL 191 is considered to be 5.0 mg/L (limit value).

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 30 October 2017 to 23 March 2018

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

14-Day Dose Range Finding study to 90-Day inhalation study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

shorter duration (14-day instead of 28-day)

GLP compliance:

no

Remarks:

Dose Range Finding study performed to determine dose levels of a subsequent 90-day study

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch no. 20170320 provided by Solvay
- Expiration date of the lot/batch: 20 March 2018
- Purity test date: 20 March 2017
- Purity: 99.66%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)

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 (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult rats, 7 weeks old at the initiation of Phase 1 and 8 weeks old at the initiation of Phase 2.
- Weight at study initiation: Phase 1: Males: 194-214 g; females: 158-163 g / Phase 2: Males: 221-246 g; females: 160-180 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet (e.g. ad libitum): 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 ad libitum.
- Water (e.g. ad libitum): tap water from the municipal supply, as for human consumption from 500 ml bottle ad libitum.
- Acclimation period: at least five days prior to involvement in the study

DETAILS OF FOOD AND WATER QUALITY: see above

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 24.5°C
- Humidity (%): 31 - 58 %
- 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: 30 October 2017 To: 27 November 2017

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

other: untreated

Mass median aerodynamic diameter (MMAD):

>= 2.61 - <= 2.87 µm

Geometric standard deviation (GSD):

1.83

Remarks on MMAD:

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L (v.s. target concentration of 5.0 mg/L);, the MMAD was 2.87 micron (GSD: 1.94).
In the Phase 2, the mean achieved atmospheric concentrations were 1.34 and 5.22 mg/L (v.s. target concentrations of 1.25 and 5.0 mg/L, respectively); the MMAD were 2.66 micron (GSD: 1.95) and 2.61 micron (GSD: 1.83), for the low and high dose, respectively.

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. Two identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric stainless steel cylinders, the inner plenum and the outer chamber with 10 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.
- 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).
- Method of conditioning air: not specified
- System of generating particulates/aerosols: 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: Test atmosphere temperature, Test atmosphere relative humidity, Test atmosphere carbon dioxide concentration and Test atmosphere oxygen concentration
were monitored during each exposure period by the TSE-DACO monitoring system. The test atmosphere temperature, relative humidity, oxygen and carbon dioxide concentration were considered to be satisfactory for this type of study.
- Air flow rate: The flow of air through each port was approximately 0.5 L/min. This flow rate is considered adequate to minimise re-breathing of the test atmosphere and maintained oxygen concentrations at greater than 19% and a carbon dioxide concentration not exceeding 1%.
- Air change rate: not specified
- 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: see section "Details on analytical verification of doses or concentrations"
- Samples taken from breathing zone: yes (see above)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Based on the preliminary data, the non-volatile content of the test item is nearly 0 after 24 hours, but the volatility is quite slow in short period, therefore 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). The actual sampling schedule employed was dependent on the required sample volume in order to obtain adequate quantities of test item (exposure period).
The average concentration of the test item in the test atmosphere during filter sampling (CAS, in mg/L) was calculated by the following formula:
CAS = M / (tS x FS)
Where
M = Mass of the active substance on the sorbent (mg)
tS = Duration of sampling (min)
FS = Sampling flow rate (L/min)
In addition, the actual concentration will bewas verified by a validated GC method (16/379-316AN). Samples of the test atmosphere were collected through a G10 filter, as described above, or similar) and analysed once in Phase 2, for each dose group. Samples were collected from a vacant animal exposure port (animals breathing zone) and at least 3 samples at each concentration level were analysed at each occasion. Samples were analysed on the day of the sampling

Duration of treatment / exposure:

In Phase 1, single male and female Wistar rats Crl:WI (Han) were exposed nose-only to fixed target aerosol concentration of 5.0 mg/L for 1.5 hour, 3 hour and 6 hours per day.
In Phase 2, the animals were exposed to an atmosphere of the test item for 3 or 6 hours per day.

Frequency of treatment:

In Phase 1, animals were exposed for 3 Days.
In Phase 2, animals were exposed for 14 Days.

Dose / conc.:

4.99 mg/L air (analytical)

Remarks:

Phase 1 animals

Dose / conc.:

1.34 mg/L air (analytical)

Remarks:

Phase 2 animals

Dose / conc.:

5.22 mg/L air (analytical)

Remarks:

Phase 2 animals

No. of animals per sex per dose:

Phase 1: 1 animal/sex/exposure duration
Phase 2: 3 animals/sex/dose/exposure duration

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The starting dose level was set based on acute toxicity data where no mortality was reported up to the highest dose of 5 mg/L air.
- Rationale for animal assignment: randomisation based on body weights
- 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:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Individual clinical observations were performed prior to exposure and once during 1.5 and 3 hour exposures and twice during the 6 hour exposure whilst the animals were still restrained. 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).
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: In Phase 1, body weights were measured daily. In Phase 2, the body weight of each animal was recorded at randomization, then on Day 1 (before the exposure), and twice a week thereafter and on Day 15 (prior to necropsy, fasted).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes : Food consumption was recorded in Phase 2 weekly and the weekly food consumption was calculated in Phase 2.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY/BLOOD CLOTTING: Yes
- Time schedule for collection of blood: Only in Phase 2: Blood samples for clinical pathology evaluation (haematology, coagulation, and clinical biochemistry) were collected immediately prior to the scheduled necropsy, by cardiac puncture under pentobarbital anaesthesia (on Day 15)
- Anaesthetic used for blood collection: Yes (pentobarbital anaesthesia)
- Animals fasted: Not specified
- How many animals: all phase 2 animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals:
- Parameters checked in table [No. 2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: Gross necropsy was performed on each animal. Terminally (Day 4 in Phase 1 and Day 15 in Phase 2), surviving animals were euthanised under pentobarbital anaesthesia by exsanguination.
In the Phase 1 animals only macroscopic observation was performed.
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.
In the Phase 2, the macroscopic examination of the tissues and organs listed in Table no. 3 were retained from all animals.

HISTOPATHOLOGY: Full histopathology was not performed.

Other examinations:

Organ weight measurements:
The following organs were weighed in all Phase 2 animals: Brain, Lungs, Adrenals, Epididymis, Spleen, Ovaries,Heart, Testes, Kidneys, Liver, Thymus, Uterus including cervix.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package of SAS 9.2 (when using Provantis).
In case of the SAS 9.2 software package (within the validated Provantis system) the following decision tree is applied automatically for statistical evaluation of continuous numeric data.
The normality and heterogeneity of variance between groups is checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests show no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test is carried out. If the obtained result is positive, Dunnett’s (Multiple Range) test is used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis is the better option when the normality and heterogeneity assumptions implicit in the tests are adequate.
If either of the Shapiro-Wilk or Levene tests show significance on the data, then the ANOVA type approach is not valid and a non-parametric analysis is required. A Kruskal-Wallis analysis of variance is used after Rank Transformation. If there is a positive result, the inter-group comparisons are performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in the body weight of males at the end of the treatment in the Low dose, Low dose 2 and High dose groups, but without dose response. The body weight gain of males was also significantly lower in the treatment groups comparing to the control, also without dose response. These differences are considered non-treatment related, but related to the restraint procedure, as the control animals were non restrained, while the treated animals spent 3 or 6 hours in restraint tube, resulting in different shape of their body weight curve

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Lower food consumption was reported in test item treated groups compared with the control. This change reflected the body weight gain differences between treated and control groups comparing to the control and are considered non-treatment related, but related to the restraint procedure.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to the controls, the following changes were observed in the mean organ weights:
The relative to brain liver weight in the males of the High Dose group (p<0.05) and in each female group was statistically significantly lower (p<0.01 in Low dose, p<0.05 in the other groups). In females, adrenal gland weight relative to bodyweight in Low dose and High dose groups (p<0.05), and the brain weight relative to bodyweight in Low dose, High dose and High dose 2 groups were statistically significantly (p<0.05) higher.
These changes were without any dose response, therefore they are considered to be incidental and non treatment related.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEC

Effect level:

> 5 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects reported up to the highest dose tested

Critical effects observed:

no

Test Atmosphere data

Actual and nominal concentrations

The exposure concentrations were monitored by gravimetrical analysis of the test item captured in glass fibre filters.

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L, the MMAD was 2.87 micron (GSD: 1.94).

The achieved test atmosphere concentrations were confirmed byan analytical methodusing a GCmethod in Phase 2. The mean achieved actual test atmosphere concentrations in Phase 2 based on the gravimetry and specific analysis are presented in the table below. 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.

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	GC analysis
			Mean of all samples	Mean of all investigated samples
2	Low Dose 1 and 2	1.25	1.3442 (SD: 0.0739)	1.1828 (SD:0.07)	5.3089 (SD:0.3978)	2.66/1.95
4	High Dose 1 and 2	5.0	5.2204 (SD: 0.1552)	4.7959 (SD: 0.17)	25.8878 (SD:1.2243)	2.61/1.83

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 5.5% and 2.97% in the Low 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 lower than gravimetry by approximately 12.0% at the Low dose level and 8.13% 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.

The particle size distribution data werewithin the range[DN1] in both phases of the study.

Based on these data the test atmosphere in each treatment group is considered respirable.

Exposure conditions

The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres.

The temperature, humidity, oxygen and carbon dioxide values 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.

Conclusions:

The exposure to the test item AUGEO SL 191 to Hannover Wistar rats for 2 weeks for 3 and 6 hours/day on a 7 day per week basis at dose levels 1.25 mg/L to 5.0 mg/L did not cause any clinical sign or any treatment related effect in the body weight, clinical pathology or histopathology. The Maximum Tolerated Dose (MTD) and the No Observed Adverse Effect Level (NOAEL) are considered to be higher than the High dose (5.0 mg/L) in this study.

Executive summary:

The objective of this 14-day study was to determine the Maximum Tolerated Concentration (MTC) and to obtain information on the toxicity of the test item when administered to Wistar (Han) rats via inhalation route for 14 days with the aim of inducing toxic effects but no death or suffering at the highest concentration, and a NOAEC at the lowest concentration level. The results of the study are for dose level selection for a subsequent 90-day inhalation toxicity study.

 

In Phase 1, single male and female Wistar rats Crl:WI (Han) were exposed nose-only to fixed target aerosol concentration of 5.0 mg/L for 1.5 hour, 3 hours and 6 hours in the first day. As no clinical signs were observed, the exposure was continued at this concentration for two more consecutive days. The objective of this phase was to find the maximum tolerated concentration (MTC) and to determine suitable concentrations for Phase 2.

Based on the results of Phase 1, two atmosphere concentrations, 1.25 and 5.0 mg/L, were set up in two inhalation systems and three male and three female Wistar rats Crl:WI (Han) were exposed to the test atmosphere in each tower for 3 and 6 hours per day on a 7 day per week basis for a period of 2 weeks (total study duration of 14 days).

A control group of three male and three females was an untreated group.

Surviving animals including controls were terminated on the day following the last exposure on Day 15.

The animals were exposed to the test atmosphere with liquid aerosol using the Test Item as supplied. The test item was generated using a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany).

 

In Phase 1, mortality, clinical observations and body weight was measured.

 

In Phase 2, 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 in Phase 1 and Phase 2 and chemical analysis in Phase 2. The results of the test atmosphere characterization were considered suitable for the study purposes.

Results

In the Phase 1, the mean achieved atmospheric concentration was 4.99 mg/L (v.s. target concentration of 5.0 mg/L); the MMAD was 2.87 micron (GSD: 1.94).

In the Phase 2, the mean achieved atmospheric concentrations were 1.34 and 5.22 mg/L (v.s. target concentrations of 1.25 and 5.0 mg/L, respectively); the MMAD were 2.66 micron (GSD: 1.95) and 2.61 micron (GSD: 1.83), for the low and high dose, respectively.

No mortality and no clinical signs observed in the study.

The body weight gain was lower in each treatment groups comparing to the control, but without dose response. It was due to the restraint and treatment procedure, as the control animals were not treated on inhalation tower.

Lower food consumption was reported in test item treated groups compared with the control. This change reflected the body weight gain differencesbetween treated and control groups and was considered non-treatment related. .

There were no treatment related changes in clinical pathology.

There was no treatment related effect in the organ weights.

There were no treatment related macroscopic observation and no effect on the respiratory tract.

 

In conclusion, the exposure to the test item AUGEO SL 191 to Hannover Wistar rats for 2 weeks for 3 and 6 hours/day on a 7 day per week basis at dose levels 1.25 mg/L to 5.0 mg/L did not cause any clinical signs or any treatment related effect in the body weight, clinical pathology or histopathology.

The MTD and the NOAEL are considered to be higher than the High dose (5.0 mg/L) in this study. As 5.0 mg/L is the maximum limit concentration in inhalation studies with liquid aerosols, no testing at higher dose levels is required.

Based on the results of the DRF study, the dose levels for the subsequent 90d GLP study should be 5.0, 1.5 and 0.5 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

5 000 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and OECD 413 compliant study.

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

Oral route:

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test conducted according to OECD Guideline 422 and in compliance with GLP, 2,2-Dimethyl-1,3-dioxolane-4-methanol diluted in Phosphate Buffer Saline solution was administered daily by oral gavage to male and female Sprague-Dawley rats (10/sex/dose), for 2 weeks before mating, during mating and (for females) throughout gestation and until day 5 post-partum, at dose-levels of 250, 500 or 1000 mg/kg bw/day. A control group was treated with Phosphate Buffer Saline solution.

No test item-related deaths or clinical signs were noted in any group and sex. No toxicologically relevant effects on mean body weight, mean food consumption, mean Functional Observation Battery and motor activity data and mean clinical pathology parameters in any group and sex. The increase in the absolute and relative liver weights recorded in males given 1000 mg/kg bw/day was considered not to be adverse in view of the slight magnitude of the changes and the absence of microscopic correlates. None of the macroscopic findings were attributed to the test item administration. At histopathology, tubular hyaline droplets in the kidneys (consistent with rat-specific alpha-2u-globulin) were seen with increased incidence and severity in males treated at 1000 mg/kg bw/day compared to controls (presence of dense eosinophilic droplets in proximal tubular epithelium). This finding was not observed at 250 or 500 mg/kg bw/day. These kidney effects were considered to be related to alpha-2u globulin nephropathy and of no relevance to humans. There were no toxicologically relevant effects on mean mating, fertility and delivery data in any group. There were no test item-related effects on pups by day 5 post partum (viability, clinical signs, sex ratio, macroscopic post-mortem findings).

 Under the test conditions, the No Observed Adverse Effect Level (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bw/day in rats (based on findings in the kidneys of males).

 

Inhalation route:

In a subchronic inhalation toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, 2,2-Dimethyl-1,3-dioxolane-4-methanol, administered to Hannover Wistar for 90 days at 0.5, 1.5 and 5.0 mg/L atmosphere concentration (mist), caused increased incidence and severity of the eosinophilic droplets in the renal cortex of the High dose males. The immunohistochemical investigation showed that these droplets contain alpha-2 microglobulin and are indicative of alpha-2 microglobulin nephropathy, a phenomenon exclusively found in adult male rats. This finding was therefore considered to be non-adverse because of no relevance to human. 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.

In conclusion, under the conditions of this sub-chronic inhalation toxicity study, the No Observed Adverse Effect Concentration (NOAEC) for AUGEO SL 191 was considered to be 5.0 mg/L (limit value).

These results were confirmed by the results of the 14 -day Dose Range Finding study aiming at determining dose levels for the 90-day study. In this study where male and female Wistar rats were exposed nose-only to two atmosphere concentrations, 1.25 and 5.0 mg/L, for 3 and 6 hours per day on a 7 day per week basis for a period of 2 weeks (total study duration of 14 days)., no mortality nor clinical signs were observed. There were no treatment-related adverse effects on body weight an food consumption. There were no treatment related changes in clinical pathology. There was no treatment related effect in the organ weights. There were no treatment related macroscopic observation and no effect on the respiratory tract. The MTD and the NOAEL was considered to be higher than the High dose (5.0 mg/L), like in the subsequent subchronic study.

Justification for classification or non-classification

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity by the oral route was considered to be the highest dose tested in rats (i.e 1000 mg/kg bw/day) based on findings in the kidneys of males which were considered not relevant to humans (consistent with rat-specific alpha-2u-globulin).

In addition, no relevant to human adverse systemic or local effects were reported in a subchronic toxicity study by the inhalation route in rats leading to a NOAEC of 5 mg/L. Indeed, the only treatment-related finding consisted in increased incidence and severity of the eosinophilic droplets in the renal cortex of the High dose males.

Immunohistochemical investigation showed that these droplets contain alpha-2 microglobulin and are indicative of alpha-2 microglobulin nephropathy, a well-known phenomenon exclusively found in adult male rats. This result is consistent with the kidney findings reported in the oral study and was therefore considered to be non-adverse because of no relevance to human.

Thus, 2,2-Dimethyl-1,3-dioxolane-4-methanol does not need to be classified for repeated dose toxicity according to the Regulation (EC) No. 1272-2008 (CLP) and GHS UN.