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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2-dimethyl-1,3-dioxolan-4-ylmethanol
EC Number:
202-888-7
EC Name:
2,2-dimethyl-1,3-dioxolan-4-ylmethanol
Cas Number:
100-79-8
Molecular formula:
C6H12O3
IUPAC Name:
(2,2-dimethyl-1,3-dioxolan-4-yl)methanol
Test material form:
liquid
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%)

Test animals

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

Administration / exposure

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).
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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

Effect levels

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)

Target system / organ toxicity

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

Any other information on results incl. tables

 

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
(% < 3
mm)

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 18.7oC). 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

9.95dd

9.80dd

10.20     9.4-11.0

differences %

-1.3

-2.5

-4.0

 

Females

 

 

Haemoglobin (g/dL)

15.29

15.10

14.70d

15.06

15.15    14.0-16.5

differences %

-1.2

-3.9

-1.5

 

MCV (fL)

56.16

56.27

57.26

57.79u

54.95    51.8-59.1

differences %

0.2

2.0

2.9

 

Basophils Rel. (%)

0.72

0.51

0.76

0.22u

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

0.252dd

0.258d

0.262d

0.332      0.10-1.09

differences %

-20.3

-18.4

-17.1

 

AST/GOT (U/L)

150.4

263.1u

270.9

180.7

169.9      101-658

differences %

74.9

80.1

20.1

 

ALT/GPT (U/L)

45.1

95.8uu

100.6u

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

8.708d

differences (%)

1.3

8.7

15.9

Liver/BW (%)

2.298

2.250

2.391

2.674uu

differences (%)

-2.1

4.1

16.4

Liver/Brain (%)

363.23

366.74

393.55

417.67d

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

0.671u

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

5.962dd

differences (%)

10.1

9.4

17.3

Liver/BW (%)

2.656

2.838

2.829

2.940dd

differences (%)

6.9

6.5

10.7

Liver/Brain (%)

263.13

286.96

280.98

295.89d

differences (%)

9.1

6.8

12.4

Kidneys (g)

1.286

1.352

1.395u

1.467uu

differences (%)

5.1

8.5

14.1

Kidneys/BW (%)

0.674

0.687

0.711

0.723d

differences (%)

1.9

5.5

7.3

Kidneys/Brain (%)

66.62

69.28

70.53

72.75dd

differences (%)

4.0

5.9

9.2

Adrenals (g)

0.0675

0.0775

0.0785u

0.0836uu

differences (%)

14.8

16.3

23.9

Adrenals/BW (%)

0.0354

0.0392

0.0400

0.0411d

differences (%)

10.5

13.0

16.1

Adrenals/Brain (%)

3.499

3.975

3.974

4.148uu

differences (%)

13.6

13.6

18.5

Brain (g)

1.931

1.952

1.979

2.016dd

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

Applicant's summary and conclusion

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).