Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 25 June 2017 to 23 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

Test material information

Constituent 1

Reference substance name:

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

EC Number:

939-368-0

Cas Number:

1322-93-6

Molecular formula:

Not applicable (a generic molecular formula can not be provided for this specific UVCB substance)

IUPAC Name:

Reaction product of naphthalene, propan-2-ol, sulfonated and neutralized by caustic soda

Test material form:

solid: particulate/powder

Details on test material:

- Storage condition of test material: Room temperature, protected from humidity

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, Sandhofer Weg 7, D-97633 Sulzfeld.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks old at the initiation of treatment
- Weight at study initiation: Males: 221-250 g; females: 156-173 g
- Fasting period before study: no
- Housing: Group caging (in groups of 3 or 2 per cage, by sex)
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 (males) - 24 (females) days

DETAILS OF FOOD AND WATER QUALITY:
- The animals were provided with ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- tap water from the municipal supply, as for human consumption from 500 ml bottle

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 – 27.9°C
- Humidity (%): 30 - 78 %
- Air changes (per hr): 15-20 air exchanges per hour.
- Photoperiod (hrs dark / hrs light): 12 hours of continuous artificial light in each twenty-four period (from 6.00 a.m. to 6.00 p.m.)

IN-LIFE DATES: From: 25 June to 30 September 2017

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 0.68 - <= 1.16 µm

Geometric standard deviation (GSD):

2.08

Remarks on MMAD:

Group 2 (Low dose): MMAD = 0.68 (GSD = 2.03)
Group 3 (Mid dose): MMAD = 0.99 (GSD = 2.08)
Group 2 (High dose): MMAD = 1.16 (GSD = 1.89)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: The animals were treated by the inhalation route using a nose-only exposure unit, in a TSE Rodent Exposure System with each individual concentration or control group in a dedicated tower. Four identical, modular multilevel flow – past, nose only exposure units (towers) were used. The exposure unit consisted of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports. The equipment was supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature/RH, O2 and CO2 sensors or other similar equipment. These units were manufactured by TSE Systems GmbH, Bad Homburg, Germany and are similar to the inhalation system evaluated by Pauluhn. The exposure units were placed in closed hoods in order to avoid cross contamination and contamination of the laboratory environment.
- Method of holding animals in test chamber: The animals were held in polycarbonate restraint tubes located around the chamber which allowed only the animals’ nares to enter the exposure port.
- Source and rate of air: Airflows and relative pressures within the system were constantly monitored and controlled by the computer system thus ensuring a uniform distribution and constant flow of fresh test atmosphere to each exposure port (breathing zone). The flow of air through each port was approximately 0.5 L/min.
- System of generating particulates/aerosols: For the test atmosphere generation, a stainless steel concentric jet nebuliser (TSE Systems GmbH, Bad Homburg, Germany) located at the top of the exposure chamber was used. The rate of formulation use was controlled by a syringe pump. Verification of the target concentration, particle size and particle distribution was measured gravimetrically. Technical trials included filter analysis. Technical trials with the aqueous formulations had shown that the droplets become a powder in the dry air, so the rats were exposed principally to a powder (samples collected at the animal breathing zone were used for atmosphere characterisation).
- Temperature, humidity, pressure in air chamber: see above ("Exposure apparatus")
- Air flow rate: approximately 0.5 L/min
- Method of particle size determination: The aerosol fraction was measured and characterized using a 7-stage cascade impactor of Mercer style (TSE Systems GmbH, Bad Homburg, Germany). Such devices employ an inertial separation technique to isolate particles into discrete aerodynamic size ranges. Samples were collected weekly at each concentration tested and measured gravimetrically. Samples were collected from a vacant animal exposure port (animals breathing zone) and the resulting data used to calculate the mass median aerodynamic diameter (MMAD), Geometric Standard Deviation (GSD) and percentage <3 μm (considered to be inhalable in the rat).
- Treatment of exhaust air: After passing through the animal’s breathing zone, spent test atmosphere entered the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany). Sampling was normally performed each day shortly after chamber equilibration and then at regular intervals during the exposure, and samples were collected from a vacant animal exposure port (animals breathing zone).
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual (achieved) concentration of generated atmospheres was measured gravimetrically at regular intervals during an exposure by pulling a suitable, known volume of test atmosphere, from the exposure chamber, through GF10 glass fibre filters (Whatman, Germany) (see results in table 5)

Duration of treatment / exposure:

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

Frequency of treatment:

6 hour per day ; 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.004 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

0.01 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.04 mg/L air (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: The dose levels were selected based on previous data available, including the results of preliminary dose range finding studies in the rat
(see 4-week studies 15/352-212PE and 15/352-212PER dated 2017-218 reported in section 7.5.2).
- Rationale for animal assignment (if not random): not applicable (animal were randomized)
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random):not applicable

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Checks for mortality and/or morbidity were made twice daily, early and late during the normal working day. As a minimumon exposure days, individual, clinical observations were performed prior to exposure and during exposure whilst the animals were still restrained (limited observation). Following exposure, clinical observation was performed at least twice (as
soon as practicable after removal from restraint, and approximately one hour after completion of the exposure). In non-treatment days, clinical observation was made once.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage in a standard arena. The first detailed observation was made on Day 8 then once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight of each animal was recorded with precision of 1 g at randomization, and twice a week thereafter (except Week 1) including Day 90 (last exposure day) and fasted on Day 91.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
Ophthalmoscopic examination was conducted in all animals before treatment and on Week 13.
Mydriasis was produced after instillation of eye drops "Cicloplegicedol" (10 mg/mL ciclopentolato hydrochloride; Batch No.: 160515, exp.: May 2021; Batch No.: 170120, exp.: January 2022) into the conjunctival sac. The evaluation was performed by external examination and using a Gowllands or Heine Omega 500 ophthalmoscope.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment period, prior to scheduled necropsy on Day 91.
- Animals fasted: Not specified
- How many animals: all animals
- Parameters checked in table [2] were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Towards the end of the treatment period on Day 77-78, each animal was subjected to the functional observation battery, including measurements of the landing foot splay, fore/hind grip strength and motor activity assessment, on one of the 2 days per week when there was no exposure.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed.
A detailed assessment for neurotoxicity effects were made on the basis of these measurements (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).
Parameters such as body position, locomotor activity, respiration rate, respiration type, piloerection, head searching, compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna reflex, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and vocalisation were evaluated.
To measure the landing foot splay, the hind paws of the rat were painted with ink and the rat was dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured. The fore paws of the rat were also painted and measured, but they were not evaluated.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively
rodent muscular strength, in order to identify and assess quantitatively any potential effect of Test Item. The rats were held appropriately such that the fore limbs are allowed
to grip the support bar and pulled back until they release the bar; the device measures the maximum grip strength. This was performed 3 times for each animal on the test day.
The procedure was repeated with the hind limbs with the appropriate grip support. The results were tabulated with individual and mean data.
Motor activity assessment was conducted using Automatic Monitoring System of rat locomotor activity SMART v. 2.5 (Harvard Apparatus, Germany). Locomotor activity was monitored by placing each animal individually into an open-field for 1-hour observation time, when DVD recording of movement was made. Recording was made for a duration of 30 min, under dim-light and undisturbed conditions. The DVD was analysed with “SMART” software after all recordings were made to produce the appropriate parameters. Data from each dose and control group were evaluated for distance travelled in 5 minute segments. The data from the 5 minute segments were presented graphically with the intention of showing plateau activity in controls, and comparing the treatment groups.


IMMUNOLOGY: No

Sacrifice and pathology:

Gross necropsy was performed on each animal. Terminally on Day 91 surviving animals were euthanized under pentobarbital anaesthesia by exsanguination.
After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.
GROSS PATHOLOGY: Yes (see table 4)
HISTOPATHOLOGY: Yes (see table 4)

Other examinations:

ORGAN WEIGHTS: (see table 3)

EXAMINATION OF VAGINAL SMEARS:
Prior to necropsy, the oestrus cycle of all females were determined by taking vaginal smears, which were prepared and stained with 1% aqueous methylene blue solution.
The smear was examined with a light microscope, in order to provide information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of oestrogen sensitive tissues.

Statistics:

Statistical analysis was performed using SAS 9.2 (built in Provantis System).
The following decision tree was automatically applied within the validated Provantis system for statistical evaluation of numeric data:
The normality and heterogeneity of variance between groups were checked by Shapiro-Wilk and Levene tests using the most appropriate data format (log-transformed when justified). Where both tests showed no significant heterogeneity, an Anova / Ancova (one-way analysis of variance) test was carried out. If the obtained result was positive, Dunett (Multiple Range) test was used to assess the significance of inter-group differences; identifying differences of <0.05 or <0.01 as appropriate. This parametric analysis it the better option when the normality and heterogeneity assumptions implicit in the tests were adequate.
If either of the Shapiro-Wilk or Levene tests showed significance on the data, then the ANOVA type approach was not valid and a non-parametric analysis was required. A Kruskal-Wallis analysis of variance was used after Rank Transformation. If there was a positive result, the inter-group comparisons was performed using Dunn test; identifying differences of <0.05 or <0.01 as appropriate.
For non-continuous data, the Cochran-Armitage test for trend was applied and the Chisquared test was used for statistical differences relative to control.
For pathology data (macroscopic and microscopic data) the Cochran-Armitage test for trend was applied, then if appropriate, the Chi-squared test homogeneity test. If significance was plausible based on a user-defined value (0.05), a pairwise test of each treatment group versus the control group was made. If the group size was <5 then Fisher’s Exact Test was used, if the group sizes were bigger then the Chi-squared test was used; identifying differences of <0.05, <0.01 or <0.001 as appropriate.

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 groups, transient, sporadic diuresis was recorded in males on Days 19, 20 and 23 and in females on 12, 13 and 16 in the High dose group. Sporadic appearance of scars was also observed in Low and High Dose male groups. These findings were not considered to be adverse effects of treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared with the control groups, there were significantly lower body weights at the end of the treatment in the Mid and High doses (9.4% and 13.1% (p<0.01) in Mid and High dose males and 5.2% (p<0.05) in High dose females) and the overall body weight gains were significantly lower in both sexes (27.3% and 37.9% in Mid and High dose males; 16.0% and 19.5% in Mid and High dose females).
It is considered that the reduced weight gain in Mid and High dose males was an adverse effect of treatment. Similarly, but to a lesser extent, High dose females were also affected; Mid dose female weight differences were similar to the High dose females, but were generally not statistically different from controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food intakes reflected the body weight gain differences, with lower food intake in the High dose males most weeks and some weeks in the Mid dose males, with the overall
food intake significantly lower than control only in High dose males. There were no statistical differences in females.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination. All examined animals were found to be normal.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the haematology parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is considered that there were
no adverse haematology changes in the study.
The WBC count for some control males was higher than typically seen; apparently statistically lower WBC values in the treated groups was not considered to be a
treatment effect, taking into account control data from a contemporaneous study. This difference consequentially caused the absolute WBC sub-populations to be statistically
different as well, but since the relative counts were clearly not affected by treatment, these differences were not of biological significance. There were no effects on female
WBC parameters.
Small differences in RBC, Hct, MCV or MCH were all close to the expected range without any consistent indication of a test item effect on erythrocyte parameters, these
were not considered to reflect an adverse effect of treatment. Similarly, the differences in APTT or PTT were relatively small and were similarly considered as sporadic
statistical differences and not an adverse effect of treatment.
None of the statistical differences in haematological parameters are considered to represent an adverse effect of the test item.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the clinical chemistry parameters, there were a few statistical differences. To establish their relationship with treatment, control data from a contemporaneous study and the data from the clinical chemistry of the preliminary study with this test item were examined. The statistical differences for Creatin were clearly without a dose response.
The statistical differences for Sodium, ALT and ALKP were close to the expected control range, did not show indications of a dose response and showed no differences in the preliminary 28-day study (Report 15/353-212PE) hence these statistical differences were not considered to represent a treatment related effect. The lower Glucose and Triglycerides in High dose males, and the apparent reductions in Protein Albumin and A/G ratio in High dose females were all changes that mirrored the changes seen in the 28-day preliminary study, hence although the magnitude of the changes were not great, they were ascribed to being an effect of treatment. In the absence of any histological changes in the liver or kidney, the toxicological significance of these changes is equivocal; the magnitude of the differences suggests these are not indicative of significant adverse systemic toxicity.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity in any dose group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The following treatment related changes were observed in the mean organ weights:
The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females. The lung weight relative to body weight was about 21% and 55% above control in Mid and High dose males respectively; in female Mid and High dose groups the differences were similar at about 24% and 58% respectively.
In the thymus (an organ generally sensitive to stress) there appeared to be a degree of individual stress related with treatment. The absolute and relative (to body and/or brain) thymus weights were statistically significantly lower in the Mid and High dose groups of males (abs. weights: 29.7% and 35.2% (p<0.01); rel. to bodyweights: 22.8% and 25.6% (p<0.01) and rel. to brain: 26.8% and 32.9% (p<0.01)) and in the High dose group of the females (abs. weight: 25.5% (p<0.05); rel. to brain: 26.8% (p<0.05)). Histopathology confirmed decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
Other statistical differences in organ weight were generally directly associated with the body weight differences, with lower absolute weights or higher relative weights of some organs, but with no relationship with systemic effects on the organs. The female adrenal (absolute and relative) appeared higher than control, but in the absence of any histopathological change this difference was not considered to be an adverse effect of treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related macroscopic changes were not seen during the necropsy.
Other findings, like thin fur, scares on the skin, enlarged mediastinal lymph node (in 1/10 High dose male), dark red discoloration of the thymus (in 1/10 Low dose male) and the dilatation of uterus could be considered as background or procedure related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium were microscopically observed in the lungs from the Mid and High dose
male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). There were no effects observed in the Low dose. The histological changes are correlated with organ weight changes. This histological change is considered to reflect a local irritation response caused by the presence of the test item in the atmosphere; the effect is considered to be an adverse effect. There were no other changes suggestive of a degenerative pathology.
Histopathology of the thymus showed decrease of size/cellularity of the thymic cortex in High dose males. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.
The decreased cellularity in the thymus and other sporadic changes, like increased cellularity in the mediastinal lymph node, inflammatory cell infiltrate in the prostate,congestion/haemorrhage in the thymus, erosion/ulceration in the skin and signs of oestrus were considered as incidental or background.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Examination of vaginal smeares: There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the oestrus phases.

Effect levels

Key result

Dose descriptor:

NOAEC

Effect level:

0.004 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.01 mg/L air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

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 anHPLC/UV method. No test item was detected in the control air. The mean achieved actual test atmosphere concentrations based on the gravimetry and specific analysis and the nominal concentration (mass of the test item dispersed into the exposure system per total air flow used for exposure) are presented in the following table.

Mean achieved actual and nominal test atmosphere concentrations

Gr. No.	Group Designation	Target Concentration (mg/L)	Achieved Concentration (mg/L)		Nominal Concentration (mg/L)	MMAD/GSD (micron)	MMAD/GSD (micron) analytical
			Gravimetry	HPLC analysis
			Mean of all samples	Mean of all investigated samples
2	Low	0.004	0.0041 (SD: 0.0001)	0.0042 (SD: 0.0003)	0.0051 (SD:0.0003)	0.61/2.91	0.68/2.03
3	Mid	0.01	0.0104 (SD: 0.0004)	0.0108 (SD: 0.0005)	0.0128 (SD:0.0009)	0.96/2.30	0.99/2.08
4	High	0.04	0.0394 (SD: 0.0018)	0.0393 (SD: 0.0015)	0.0489 (SD:0.0024)	1.16/2.09	1.16/1.89

 

The feed rate of the test item was adjusted during the study according to the result of the gravimetric and analytical measurements at least daily, in order to achieve the target concentration. As a result, the mean deviation from the gravimetric concentration were 2.4% (max. of 7.3%), 3.8% (max. of 13.5%) and 4.6% (max. of 26.6 %) in the Low, Mid and High doses, respectively. These stability values were considered to be adequate for the study purposes. The average concentrations achieved were close to the target concentration.

Results of the analytical measurements were slightly higher than gravimetry by approximately 2.4% and 3.8% at the low and mid dose levels and slightly lower by 0.25% at the high dose level.

Nominal concentrations were higher than actual measured concentrations; this is normal since a fraction of the test item always accumulates in the separators, tubing or in the towers.

Particle size analysis

According to the results, the Mass Median Aerodynamic Diameter of the test atmospheres of all groups was in the range of 0.61-1.16 µm with Geometric Standard Deviation of 2.09-2.91.

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

Particle size distribution data of the aerosol fraction (MMAD and GSD):

Group	Mean Mass Median Aerodynamic Diameter (MMAD) (mm)	Geometric Standard Deviation (GSD)	Inhalable Fraction (% < 3mm)
2	0.61	2.91	94.7
3	0.96	2.30	91.5
4	1.16	2.09	90.2

 

Exposure conditions

The airflow through the chamber was adequate to keep dynamic flow and to avoid re-breathing of the test atmospheres. Temperature of the test atmospheres and air were within the range during the study. The relative humidity was occasionally out of the optimal range of 30-70% or unmeasurable due to the use of water formulation for the dispersion of the test item. This deviation had no effect on the purpose and integrity of the study.

The oxygen and carbon dioxide concentrations were considered to be satisfactory for this type of study.

In conclusion, the results of the atmosphere characterization exhibited that the test atmospheres were suitable for the purposes of the study.

Applicant's summary and conclusion

Conclusions:

Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused decreased
body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item.
There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.
In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m3).

Executive summary:

The objective of this 90-day study was to evaluate the potential toxic effects of the test item, Supragil WP, when administered to Wistar (Han) rats via inhalation route for at least 90 days. This study was carried out according to OECD test guideline No. 413 (September 2009), in compliance with the Good Laboratory Practices (GLP).

Three groups of 10 male and 10 female Wistar rats Crl:WI (Han) were exposed to the test item, at target concentrations of 0.004, 0.01 and 0.04 mg/L using a nose-only

exposure system. A vehicle control group of 10 male and 10 female rats was exposed to distilled water. The animals were exposed to the test atmosphere for 6 hour per day on a 5 day per week basis for a period of 13 weeks (total study duration of at least 90 days). Start of the exposure for all 4 groups was Day 1.

The treatment was performed in 4 inhalation towers in parallel, one tower for each treatment groups and an additional tower for the vehicle control group.

Surviving animals including controls were terminated on the day following the last exposure on Day 91.

The achieved concentrations and particle size distributions for all groups were considered to be fully acceptable for Concentration and Stability of the exposures.

Analysis of filter samples for concentration were performed and no Test Item was detected in the control samples.

Results:

There was no mortality in the study.

No treatment related clinical signs were observed in the treatment groups.

There was no effect in the Grip Strength, Landing Foot Splay, Irwin Test or locomotor activity; hence no neurological effects were identified in any dose group.

There were significantly lower body weights at the end of the treatment in the Mid and High dose groups and the body weight gains were significantly lower in both sexes, particularly in males. Food intakes reflected the body weight gain differences.

No Test Item related changes compared to pre-treatment or/and the Control were noted at ophthalmoscopy examination.

In the haematology and clinical chemistry parameters, there were a few statistical differences, but not consistent between sexes or clearly related to treatment. It is

considered that there were no adverse clinical pathology changes in the study.

There were no adverse or Test Item related observations in the animal oestrus cycle evaluated prior to necropsy and the animals showed the normal distribution of the

oestrus phases.

The absolute and/or relative (to body and brain) lung weights were statistically significantly higher in the Mid and/or High dose groups both in the males and females.

These changes correlated with a local irritation response of hyperplasia of the mucous cells of the bronchial/bronchiolar epithelium observed microscopically in the lungs from the Mid and High dose male and female groups (minimal severity in 8/10 Mid dose males and 6/10 Mid dose females, slight to severe in all of the High dose males and females). These changes were considered to be adverse effects of the test item.

An apparent stress effect was seen in thymus weights on some Mid and High dose males correlated, at histology, with a decrease of size/cellularity of the thymic cortex. Since there was no lymphocyte depletion of the spleen and lymph nodes identified by histopathology, thymic differences did not suggest any primary effects of the test item on the immune system.

There were no other changes in organ weights, necropsy or histological observations.

In summary, Supragil WP administered via inhalation route to Hannover Wistar rats for 90 days at 0.004, 0.01 and 0.04 mg/L atmosphere concentration, caused

decreased body weight gain and food consumption at the Mid and High doses. Increased lung weights were associated with minimal to severe hyperplasia of the mucous cells in the Mid and High dose groups. These changes were considered to be adverse effects of the test item. There were no other adverse effects of treatment in clinical signs, ophthalmoscopy, neurological assessments, clinical pathology, oestrus cycle or in other tissues or organs of treated animals. There was some evidence for decreased thymus weight in the Mid and/or High dose male groups, considered as stress-related rather than a direct effect, confirmed at histology.

In conclusion, under the conditions of this inhalation study, the no observed adverse effect concentration (NOAEC) for Supragil WP is considered to be 0.004 mg/L (4 mg/m³).