Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Endpoint summary

Description of key information

The substance was evaluated in three repeated-dose toxicity studies performed by the oral and inhalation routes. By the oral route, rats treated for 14 days showed no remarkable effects up to a dose-level of 1000 mg/kg bw/day. By inhalation, no systemic effects were observed in rats exposed up to 2020 mg/m3 air for 14 days or up to 103.4 mg/m3 air for 90 days while local respiratory effects were noted. Histopathological evaluation performed in the subchronic study revealed local pulmonary effects. The cellular changes in the bronchoalveolar lavage (BAL) and haematology  were consistent with the microscopic findings and confirmed the local pulmonary toxicity of the test substance. Based on these findings, the NOAEC was set to 3.06 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

other: range-finding study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From 14 August 2012 to 19 November 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

The objective of this study was to evaluate the potential toxicity of the test substance following daily oral administration (gavage) to Crl:CD(SD) rats for 14 days, in order to select the dose-levels for an OECD 421 reproductive/developmental toxicity screening study.

GLP compliance:

no

Remarks:

Range -finding toxicity study

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 10 weeks
- Weight at study initiation: 353 to 394 g (males) and 234 to 263 (females)
- Fasting period before study: no
- Housing: Four of one sex per cage. The cages were made of a polycarbonate body with a stainless steel mesh lid.
- Diet: Ad libitum, standard rodent diet (SDS VRF1 Certified). This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr): Each animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated
- Photoperiod (hrs dark / hrs light): 12 h continuous light and 12 h continuous dark/24 h.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): homogeneous and stable suspensions were obtained with corn oil as a vehicle
- Concentration in vehicle: 20, 60 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg bw

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

No test substance formulation analysis was performed in this study. However, the homogeneity and stability of the formulation were determined. Formulations were confirmed stable for up to 24 hours at ambient temperature (nominally 21°C) and fifteen days when refrigerated (nominally 4°C).

Duration of treatment / exposure:

14 days

Frequency of treatment:

7 days/week

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The high dose (1000 mg/kg bw/day) for this study was the maximum dose level required for the subsequent main study (reproductive/developmental toxicity screening study). The low (100 mg/kg bw/day) and intermediate (300 mg/kg bw/day) dose levels were chosen to allow determination of a dose response.

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- A detailed physical examination was performed on Days 1, 4, 8, 11 and 15 for each animal to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded during acclimatization, on Days -3, 1, 4, 8, 11 and 15 (before necropsy).

FOOD CONSUMPTION :
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of the food supplied to each cage, food remaining and an estimate of any spillage was recorded at Day -3 to -1, 1 to 3, 4 to 7, 8 to 10 and 11 to 14. From these records the mean daily consumption per animal (g/animal/d) was calculated for each cage.

WATER CONSUMPTION: Yes
-Daily by visual observation.

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. After ventral mid-line incision, the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera were exposed and examined in situ. Any abnormal position, morphology or interaction was recorded.

HISTOPATHOLOGY:
Testes were fixed in modified Davidson's fluid. Epididymides, ovaries, kidneys, spleen and liver from all animals were preserved in 10% neutral buffered formalin but no microscopic examinations of these tissues were performed.

Other examinations:

Yes
The following organs were dissected free of adjacent fat and other contiguous tissue and the weights were recorded: epididymides, ovaries, kidneys, spleen, liver, testes, bilateral organs were weighed individually.

Statistics:

none

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):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY:
There were no premature deaths and no clinical signs.

BODY WEIGHT AND WEIGHT GAIN:
Mean bodyweight gain for females at 1,000 mg/kg bw/day was markedly lower than those at 100 or 300 mg/kg bw/day over the 2 week treatment period. However, this difference was mainly due to one female which showed overall slight bodyweight loss.The weight gain of the other 3 females was unremarkable.The overall bodyweight gain in males was similar in all groups.

FOOD CONSUMPTION:
Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females.

WATER CONSUMPTION:
The visual assessment of water intake did not reveal any dose-related effect up to the end of treatment.

ORGAN WEIGHTS:
Liver weights of females at 1000 mg/kg/day were slightly lower than those at 100 or 300 mg/kg bw/day groups. However, due to the absence of any similar finding in the males, the significance of this difference was unclear.

GROSS PATHOLOGY:
No effects

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Except a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed.

Key result

Critical effects observed:

no

Conclusions:

With the exception of a slightly lower liver weight in females at 1,000 mg/kg bw/day when compared to controls, no other effects were observed when the test substance was administered by gavage for fourteen days to male and female rats.

Executive summary:

In a dose range-finding study performed in compliance with Good Laboratory Practice, the test substance was administered daily by gavage to CD rats for 14 days.

Three groups, each comprising four male and four female rats, received the test substance at doses of 100, 300 or 1,000 mg/kg bw/day. During the study, the animals were checked at least twice daily for mortality and clinical condition. Bodyweight and food consumption were recorded twice weekly. Water consumption was assessed by daily visual observation. Animals were sacrificed on completion of the treatment period (day 15) and a complete macroscopic post-mortem examination was performed. The kidneys, liver , spleen, ovaries, testes and epididymides were weighed and preserved although no microscopic examination was performed.

There were no mortalities and the clinical condition of the animals was unaffected by treatment. Three females receiving 1000 mg/kg/day showed unremarkable bodyweight gains, but one female showed overall slight weight loss of uncertain relationship to the treatment. Overall bodyweight gain in males was similar in all groups. Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females. There were no effects of treatment on the organ weights of animals which received 100, 300 or 1000 mg/kg/day except for a slightly lower liver weight in females that received 1,000 mg/kg bw/day. Macroscopic examination at necropsy after 14 days of treatment did not reveal any abnormalities.  

Based on these results, it could be concluded that the NOAEL for the test substance was 1000 mg/kg bw/day and dose levels of 100, 300 and 1000 mg/kg/day would be suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.

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:

Dose Range-Finding study with a limited number of parameters assessed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 November 2012 to 14 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

This pilot study was performed in order to obtain preliminary information about the local and systemic toxicity of the substance when administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats. The results of the study are intended to be used for dose level selection for the 90-day inhalation study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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
- Age at study initiation: Young adult rats, 9 weeks old
- Weight at study initiation: 171 to 299 g (males: 268-299 g; females: 171-207g)
- Housing: group caging (5 animals/sex/cage)
- Bedding: Lignocel bedding for laboratory animals was available to animals during the study
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity: 30–70 %
- Air changes: 15-20 air exchanges/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Deviation:The temperature (22±3ºC) and the humidity (30–70%) values deviated from the required range during the acclimation period in the animal room. However, these deviations had no effect on the purpose and integrity of the study.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.97 - <= 2.73 µm

Geometric standard deviation (GSD):

2.9

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 1.97-2.73 µm with Geometric Standard Deviation (GSD) of 2.90-3.22. Due to the test item physical properties the Geometric Standard Deviation (GSD) of the test atmosphere was slightly above 3, but was kept in range of 3±10%.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany) which is a flow-past, nose-only exposure unit. This system consists of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.The equipment is supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature, O2 and CO2 sensors.

- Method of holding animals in test chamber: animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.

- Source of air: compressed air

- Method of conditioning air: the compressed air was passed through a respiratory quality filter train and condensate separator prior to use.

- System of generating particulates: rotating brush powder disperser (Palas GmbH, Karlsruhe, Germany) located at the top of the exposure chamber

- Temperature, humidity, pressure in air chamber: 22±3 °C, 30–70 % and pressure in air chamber not reported

- Air flow rate: the flow of air through each port was at least 0.5 L/min.

- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor of Mercer sty le (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated. From this data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 3 µm was determined.

- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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, ref. no. 10370302). Sampling was normally performed shortly after chamber equilibration and then at regular intervals (approximately hourly intervals) during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure concentrations were monitored intermittently by gravimetrical analysis of the test item deposited on a sampling filter. Sometimes, marked fluctuations in sample concentration occurred. These fluctuations can be explained by the short duration of the sampling, however the average concentration was equal to the target concentrations. The nominal concentration (mass of the test item dispersed into the exposure system in total air flow used for exposure) deviates significantly from mean achieved concentration due to loss of particles in pre-separation devices used for particle size optimization.
The mean achieved concentrations were 0.10; 0.50 and 2.02 mg/L and corresponded to 100; 100 and 101% of the target concentrations respectively.

Duration of treatment / exposure:

14 days

Frequency of treatment:

5 days/ week

Dose / conc.:

100 mg/m³ air (analytical)

Remarks:

Achieved concentration for low-concentration group

Dose / conc.:

500 mg/m³ air (analytical)

Remarks:

Achieved concentration for mid-concentration group

Dose / conc.:

2 020 mg/m³ air (analytical)

Remarks:

Achieved concentration for high-concentration group

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The target concentration levels were 0.10, 0.50 and 2.0 mg/L of test item. They were set on the basis of available data and information from previous experimental work, including the results of an acute inhalation toxicity study (see section 7.2.2). In this 4–hour exposure study, no death occurred in group of six male and female rats exposed to a mean achieved atmosphere of 5.05 mg/l.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed prior to exposure on the working days, and at least twice during the 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. During the weekend (no exposure) the clinical signs were recorded once per day only.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage weekly, on Day 1, 7, 14.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with a precision of 1 g at randomization, then on Day 1 and at least on Days 4, 8, 11, 14 and 15 (prior to necropsy, fasted before the scheduled euthanasia).

FOOD CONSUMPTION: Yes
- Food was recorded on Day 1 and at least on Days 4, 8, 11 and 14. The remaining, non-consumed food given was weighed with a precision of 1g for each cage. The mean individual daily food consumption was calculated per animal.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy and macroscopic examination were performed on all animals. After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormalities were recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY: Yes
Initial processing was limited to larynx, trachea, lungs, tracheobronchial lymph nodes, nasal cavity and any gross abnormalities for all animals of all groups.

Other examinations:

Yes
The following organs were weighed : Brain, heart, kidneys, liver, lungs, spleen, testes, thymus, adrenals and ovaries.

Statistics:

The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normally distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

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, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no mortality observed during the study.
In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
• A slight increase in respiratory rate was observed in 4 out of 5 females on Day 1 and in 2 out of 5 males on Days 3-4,
• Slight and on a few occasions moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3 out of 5 animals,
• Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
• Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
• Sneezing was noted for 2 females on Days 10-11.

In the animals exposed to the concentrations of 0.50 mg/L, no significant clinical signs were noted except for 1 male where initially slight subcutaneous mass in the left axillary was recorded from Day 5 which continuously grew until the end of the study.

In the animals exposed to the concentrations of 0.10 mg/L, slightly laboured respiration was noted in 1 female on day 5. No significant clinical signs were noted for the other animals in this group during the exposure period.

No clinical signs were recorded for the air control animals.


BODY WEIGHT AND WEIGHT GAIN
In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain during the overall period of treatment (-36% compared with controls).

No effects on bodyweight and bodyweight gain were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

FOOD CONSUMPTION
In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

No effects on food consumption were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

HAEMATOLOGY
An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slightly decreased of Red Blood Count anda slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

CLINICAL CHEMISTRY

There was no effect of treatment on clinical chemistry.

ORGAN WEIGHTS
The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

GROSS PATHOLOGY
There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5 out of 5 males and 5 out of 5 females in the 2.02 mg/L group. Pale foci were seen in 2 out of 5 females of the 0.50 mg/L group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized by the presence of white/pink foreign material in the alveoli which was granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of a mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was shown by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderate at 2.02 mg/L (see table 7.5.2/3).

Key result

Dose descriptor:

LOAEC

Effect level:

100 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At 100 mg/m3, slight increase in neutrophils and slight concomitant decrease in lymphocytes were observed during the Hematology analysis. These effects were associated with a slight inflammation process in the respiratory tract of the animals

Critical effects observed:

no

Table 7.5.2/1 Mean values in affected haematologic parameters

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
White Blood Count – WBC (K/mL)	2.776	3.244	3.664	3.314	NS
Red Blood Count – RBC (M/mL)	8.726	8.384	8.272	8.096*	DN
Neutrophils –NEU, Relative (%)	25.160	21.480	28.320	34.720**	DN
Lymphocytes – LYMPH, Relative (%)	69.620	74.080	66.780	59.040**	DN
Mean Cell Haemoglobin – MCH, pg	18.18	19.00**	19.12**	19.06**	DN
Females
White Blood Count – WBC (K/mL)	1.216	2.394*	2.130*	1.948*	U
Red Blood Count – RBC (M/mL)	7.932	8.108	7.986	8.034	NS
Neutrophils –NEU, Relative (%)	14.660	20.920*	20.420*	26.360**	DN
Lymphocytes – LYMPH, Relative (%)	80.460	73.920*	74.480*	67.900**	DN
Mean Cell Haemoglobin – MCH, pg	18.94	19.32	19.18	19.62	NS

NS = Not Significant

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.2/2 Mean values of lungs weights following the 14-day exposure period

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
Lungs Weight (g)Absolute	1.318	1.620**	2.092**	2.884**	U
Relative to Brain Weight (%)	69.52	83.78**	109.68**	145.41**	U
Relative to Body Weight (%)	0.44	0.54**	0.68**	0.99**	DN
Females
Lungs Weight (g)Absolute	1.014	1.286**	1.804**	2.256**	DN
Relative to Brain Weight (%)	55.01	73.04**	98.92**	124.25**	DN
Relative to Body Weight (%)	0.51	0.64**	0.88**	1.17**	DN

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.3/3 Summary of histopatology data for lungs and tracheobronchial lymph nodes

Sex	male	male	male	male	female	female	female	female
Concentration (mg/l)	0	0.10	0.50	2.02	0	0.10	0.50	2.02
Number of Animals on Study	5	5	5	5	5	5	5	5
LUNGS;
Examined	(5)	(5)	(5)	(5)	(5)	(5)	(5)	(5)
WithinLimits	5	0	0	0	5	0	0	0
Foreign Material; alveolar; all lobes, Glanular, white/pink	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
minimal	0	4	1	0	0	5	0	0
mild	0	1	4	0	0	0	3	0
Foreign Material; alveolar; all lobes, Glanular, white/pink; correlated with necropsy.	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
mild	0	0	0	1	0	0	2	2
moderate	0	0	0	4	0	0	0	3
Inflammation, Bronchioloalveolar; mixed; all lobes	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
minimal	0	0	5	0	0	0	5	0
mild	0	0	0	4	0	0	0	1
moderate	0	0	0	1	0	0	0	1
Inflammation, Bronchioloalveolar; mixed; all lobes correlated with necropsy	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
mild	0	0	0	4	0	0	0	3
Microgranuloma; alveolar; multifocal	(0)	(0)	(0)	(3)	(0)	(0)	(0)	(3)
minimal	0	0	0	1	0	0	0	2
mild	0	0	0	2	0	0	0	1
Cell Infiltrate, Mixed Cellular; bronchiole; alveolus, all lobes	(0)	(5)	(0)	(0)	(0)	(5)	(0)	(0)
minimal	0	3	0	0	0	5	0	0
mild	0	2	0	0	0	0	0	0
LYMPH NODE, LUNG-ASSOCIATED;
Examined	(5)	(5)	(5)	(5)	(4)	(5)	(5)	(5)
WithinLimits	5	0	0	0	4	2	0	0
Not Examined: NOT PRESENT	0	0	0	0	1	0	0	0
Accumulation, Foamy Macrophage; medulla; paracortex	(0)	(5)	(5)	(5)	(0)	(3)	(5)	(5)
minimal	0	2	1	0	0	3	2	0
mild	0	3	4	0	0	0	3	1
moderate	0	0	0	5	0	0	0	4

Conclusions:

Exposure to the test substance at gravimetrically determined dose levels of 100, 500 and 2020 mg/m3 air resulted in findings in the lungs and tracheobronchial lymph nodes. In the lungs, they consisted of inflammatory reactions ranging from minimal to moderate severity, with the presence of a mixed cell population. Also, foreign material was seen in the alveoli. The incidence and severity of the inflammatory responses and the presence of foreign alveolar material were generally increased consistently to dose. A similar pattern of dose relationship was recorded for the accumulation of macrophages in the tracheobronchial lymph nodes. The low severity (minimal) in Low dose, increased (mild) in Mid dose and was moderate in High dose animals.

Executive summary:

In a pilot study performed in compliance with Good Laboratory Practice, the test substance was administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats.

The test item was administered as a dry aerosol. The animals were exposed on 10 occasions during a 14 day period. Three groups, each comprising five male and five female rats received the test substance at achieved exposure levels of of 100, 500 and 2020 mg/m3. A similarly constituted Control group received air with the same operating conditions as the other groups.

The mortality observations were performed twice daily. The general clinical observations were recorded five times for the exposed animals on the days of exposure. Detailed clinical examination was performed on Days 1, 7 and 14 prior to the 6 hour exposure. Body weight and food consumption were measured on Days 1, 4, 8, 11 and 14. Blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy one day after the last treatment. Selected organs were weighed. Histopathology investigation was performed on selected tissues.

The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3 µm) for a repeat dose inhalation study. 

There was no mortality during the study. Clinical signs related to treatment were noted only in animals given 2020 mg/m3. All but one female and only 2 males had a transient slight increase in respiratory rate. In females, slight and in few occasions moderate laboured and/ or noisy respiration was observed throughout the study. In males, slightly laboured respiration was noted only on day 2 in 3 animals. Hunched posture was observed from day 4 to day 10 in 1 to 3 females and on days 10-11 in 1 male. In addition, sneezing was noted for two females on Day 10 and 11. 

Changes in bodyweight and bodyweight gain were noted only in animals that received 2020 mg/m3. Temporary mean bodyweight loss was observed at the beginning of the study in both sexes on (Days 1-4) ( -2.2% and -0.43% in males and females respectively). This bodyweight loss correlated with the significant decrease in food consumption noted during the same treatment-period. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Following 2 weeks of treatment, an increase in White Blood Count was observed in both sexes at all doses but without dose-dependency. In addition, statistically significant increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed in males given 2020 mg/m3 and in all treated females. These changes in haematology might be contributed to changes in the lungs. A slight decrease in Red Blood Count and a slight increase of Mean Cell Haemoglobin were also observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered to be toxicologically significant. There was no effect of treatment noted during the evaluation of the clinical chemistry parameters.

Macroscopic examination revealed enlargement and/or pale discoloration of the lungs in animals given 500 or 2020 mg/m3. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels. There was a clear relationship between these microscopic findings and the dose. The alterations in the lungs were characterized by the presence of white/pink foreign material in the alveoli. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose. The inflammatory reactions in the lungs ranged from minimal to moderate severity, with the presence of a mixed cell population in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently with dose. The formation of microgranulomas was observed microscopically in the alveoli of animals treated at a dose level of 2020 mg/m3. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in the Low dose group, increased (mild) in the Mid dose and moderate in the High dose group. The mean absolute and relative lung weights adjusted for both brain and terminal body weight were significantly higher in males and females at dose levels of 100, 500 and 2020 mg/m3 compared to the controls.

Based on these findings, it was anticipated that a target maximum exposure level of 100 mg/m3 would be tolerated for a 90 day exposure in the same species.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Feb 2017 to 4 Aug 2017

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)

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI(Han))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: ±20% of the mean weight for each sex which is 231 gr for males and 169 gr for females
- Housing: Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Envirodri,Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. Animals were housed in groups of five of the same sex. Upon reaching a size at which five/cage housing was no longer allowed, male animals were housed two or three per cage. During exposure, the animals were kept individually in the exposure unit
- Diet: cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDSSpecial Diets Services, Witham, England) ad libitum.
- Water: Tap-water suitable for human consumption ad libitum
- Acclimation period: 13-16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.15 - <= 2.42 µm

Geometric standard deviation (GSD):

1.82

Remarks on MMAD:

- The average Mass Median Aerodynamic Diameter (MMAD) of the particles in the test atmosphere was 2.38 (with a geometric standard deviation of the size distribution of 1.74), 2.42 (gsd of 1.82) and 2.15 µm (gsd of 1.84) for the low-, mid- and high-concentration groups, respectively.
- Particle size distribution measurements was carried out using a 10-stage cascade impactor at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom).
- Method of holding animals in test chamber: animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated lab room different than the room where the animals were housed. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
- Source and rate of air: The average total air flows (± standard deviation) were 48.3 (± 0.3), 46.1 (± 0.1), 48.1 (± 2.0) and 66.6 (± 0.0) L/min for the control, low-, mid- and high-concentration groups, respectively
- - System of generating particulates/aerosols: The test atmosphere was generated using a turntable dust feeder (Reist and Taylor, 2000) and an eductor (Fox Valve Development Corp., Dover, NJ, USA; Cheng et al., 1989). The test material was aerosolized in the eductor, which was supplied with a flow (controlled using a reducing valve) of humidified compressed air. The resulting aerosol was led through a glass cyclone, which was used to remove the largest particles from the aerosol, and was subsequently introduced at the top inlet of the exposure chamber. The eductors were calibrated by measuring the total air flow at a range of driving air pressures encompassing the driving pressures used during the study. The driving air pressure was used to monitor the flow. A bypass stream of humidified compressed air (measured by mass view meter; Bronkhorst Hi Tec, Ruurlo, The Netherlands) was added at the top of the exposure chamber to ensure a relative humidity above 30% (for groups 2 and 3 only; this bypass flow of humidified air was not necessary for group 4). The resulting test atmosphere was directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted.
- Temperature, humidity, pressure in air chamber: The average temperature (± standard deviation) was 22.5 (± 0.4), 22.9 (± 0.4), 23.0 (± 0.4) and 23.4 (± 0.4)°C for the control, low-, mid- and high-concentration groups. The average relative humidity during exposure was 42.6 (± 1.4), 41.6 (± 2.8), 41.4 (± 2.1) and 40.2 (± 1.2) % for the control, low-, mid- and high-concentration groups, respectively.
- Air flow rate: The average total air flows (± standard deviation) were 48.3 (± 0.3), 46.1 (± 0.1), 48.1 (± 2.0) and 66.6 (± 0.0) L/min for the control, low-, mid- and high-concentration groups
- Method of particle size determination: using a 10-stage cascade impactor (2110k cascade impactor, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition.


TEST ATMOSPHERE
- Samples taken from breathing zone: test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) samples of test atmosphere through fibre glass filters (Sartorius 13400-47, Ø 47 mm) at a flow of 9.2 (group 2) or 4.6 (groups 3 and 4) Ln/min
- The chamber air flow was recorded at least hourly during exposure by recording the readings of the mass view meters and driving air pressure of the eductors.
- Temperature and the relative humidity of the test atmospheres were measured continuously during exposure, and recorded on a PC every minute using a CAN transmitter with temperature and relative humidity probes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric analysis
The actual concentration of the test material in the atmosphere was measured at least three times during exposure on each exposure day by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) samples of test atmosphere through fibre glass filters (Sartorius 13400-47, Ø 47 mm) at a flow of 9.2 (group 2) or 4.6 (groups 3 and 4) Ln/min. Samples with a volume of 1002.4, 151.8 or 32.2 Ln were taken for group 2, 3 and 4, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and then weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.

Chemical analysis
The content of the test material captured on the gravimetric filters was determined five times during the study by chemical analysis. Samples of the low-, mid- and high-concentrati n test atmosphere (and one blank) obtained during exposure of the animals in week 1, 4, 7, 10 and 13 were analyzed. The content of the test item on the filters was determined using ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS), using the following three main test substance constituents as markers to check whether the composition of the test material in the test atmospheres was similar to the original material and stable across the exposure period.

Duration of treatment / exposure:

13 weeks (ie 65 exposure days in total) followed by a 13- week recovery period.

Frequency of treatment:

6 hrs/day, 5 days / week.

Dose / conc.:

3.06 mg/m³ air (analytical)

Remarks:

Low concentration

Dose / conc.:

20.5 mg/m³ air (analytical)

Remarks:

Mid concentration

Dose / conc.:

103.4 mg/m³ air (analytical)

Remarks:

High concentration

No. of animals per sex per dose:

10 in both the main and recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target concentrations were selected on the basis of the results of the two-week repeated inhalation toxicity study conducted in rats at exposure levels 0, 0.10, 0.50 and 2.02 mg/L (Nagy, 2013b). No animals died during the treatment period. At 2.02 mg/L, animals showed an increase in respiratory rate, laboured and/or noisy respiration. Reduced food consumption as well as reduced body weight gain and body weight loss were also observed. At 0.10 and 0.50 mg/L, there were no treatment-related clinical sings and no changes in body weight or food consumption. Treatment-related histopathological findings in the lungs and tracheobronchial lymph nodes were evident at all dose levels, however, the severity of the findings for animals that received 0.50 or 2.02 mg/L precluded these levels being selected for this study as it was unlikely that these concentrations would be tolerated for 13 weeks. As the findings in the lung were of lower severity at 0.10 mg/L, it was anticipated that a target exposure level of 100 mg/m3 will be tolerated for 13 weeks. To explore any possible concentration-response relationship, the target exposure levels for the mid- and low-concentration groups were selected at 21 and 3 mg/m3, respectively.
- Species / strain selection: The Wistar strain will be used because it is routinely used at the test facility for this type of studies.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL SIGNS
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. Clinical signs for respiration, behavior, head, perineum, skin/fur, eyes, nose, mouth, ears, extremities (legs), tail, abdomen, testes, penis, urethra, urine, faeces, injection site and general signs were observed. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, reactions to treatment and mortality were recorded.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -8 to -6) and towards the end of the exposure period in the animals of the control and high concentration groups (on days 86 to 88). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

BODY WEIGHTS
The body weight of each animal was recorded once before the start of the exposure period: on day -4 or -5 for males, and on day -6 or -7 for females. These weights were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (day 0), and twice a week thereafter. Since no exposure-related effects were seen during the first 4 weeks of the study, body weight measurements were thereafter continued on a once a week basis for the remainder of the study duration. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION
Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured from day 0 over successive periods of 7 days, until the last week before sacrifice (i.e. including the recovery period) in which food consumption was measured over a 6-day period.

HAEMATOLOGY
Haematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anesthesia at sacrifice. EDTA was used as anticoagulant, except for PT where citrate was used. The samples were discarded after analysis. In each sample the following determinations were carried out:
- haemoglobin (Hb)
- packed cell volume (PCV)
- red blood cell count (RBC)
- reticulocytes
- total white blood cell count (WBC)
- differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes)
- prothrombin time (PT)
- thrombocyte count (platelet count)
The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin (MCH)
- mean corpuscular hemoglobin concentration (MCHC)
Since (possible) exposure-related changes were observed in animals of the main groups, investigation of total and differential white blood cell counts was extended to animals of the recovery groups.

CLINICAL CHEMISTRY
Clinical chemistry was conducted at the end of the treatment period on all surviving rats after overnight fasting, at the same time blood samples for hematology were collected. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18°C) to enable reanalysis if necessary and then discarded. The following measurements were made in the plasma:
- alkaline phosphatase activity (ALP)
- bilirubin total
- aspartate aminotransferase activity (ASAT)
- cholesterol
- alanine aminotransferase activity (ALAT)
- triglycerides
- gamma glutamyl transferase activity (GGT)
- phospholipids
- total protein
- calcium (Ca)
- albumin
- sodium (Na)
- ratio albumin to globulin
- potassium (K)
- urea
- chloride (Cl)
- creatinine
- inorganic phosphate
- fasting glucose
Since no exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was not extended to the animals of the recovery groups.

Sacrifice and pathology:

SACRIFICE AND MACROSCOPIC EXAMINATION
Surviving animals of the main groups were sacrificed at the end of the exposure period (day 91) in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 3-month recovery period (day 175). The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on the animal that was killed in moribund.
At scheduled sacrifice, the following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg bw) were calculated from the absolute organ weight and the terminal body weight:
- adrenals
- brain
- heart
- kidneys
- liver
- lungs
- ovaries
- spleen
- testes
- thymus
- uterus

HISTOPATHOLOGY
For histopathological examination, samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The left lung lobes (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- adrenals
- aorta
- axillary lymph nodes
- brain (Three levels were examined microscopically (brain stem, cerebrum, cerebellum)
- cecum
- colon
- epididymides
- eyes (with optic nerve)
- exorbital lachrymal glands
- femur with joint
- heart
- kidneys
- liver
- lungs/trachea /larynx (The left lung lobe was examined microscopically at three levels, Three levels were examined microscopically (including a longitudinal section through the carina of the bifurcation) and Three levels (one including the base of the epiglottis) were examined microscopically)
- mammary glands (females)
- cervical lymph nodes
- nasopharyngeal tissue (with teeth) Six levels were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue.
- nerve peripheral (sciatic nerve)
- esophagus
- olfactory bulb
- ovaries
- pancreas
- parathyroids
- pharynx
- parotid salivary glands
- pituitary gland
- prostate
- rectum
- seminal vesicles with coagulating
- glands
- skeletal muscle (thigh)
- skin (flank)
- small intestines (duodenum, ileum and jejunum)
- spinal cord (Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
- spleen
- sternum with bone marrow
- stomach (Non-glandular and glandular parts were examined microscopically)
- sublingual salivary glands
- submaxillary salivary glands
- testes
- thymus
- thyroid
- tongue
- tracheobronchial (mediastinal)
- lymph nodes
- ureter
- urethra
- urinary bladder
- uterus (with cervix)
- all gross lesions

Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration main groups (groups 2 and 3) and the recovery groups were not processed.
Bone marrow: Bone marrow smears were prepared of all surviving animals of the main and recovery groups for possible future examination.
Histopathological examination: All preserved tissues of all animals of the control and high-concentration main groups and of the animal which was sacrificed in moribund condition, were examined histopathologically (by light microscopy). In addition, the respiratory tract (nasopharyngeal tissues, larynx, trachea and left lung lobe) and tracheobronchial lymph nodes of animals of the intermediate concentration groups of the main study were examined histopathologically. Furthermore, all gross lesions observed in rats of the intermediate concentration groups and animals of the recovery groups were examined microscopically. Histopathology will be subjected to a peer review system. Since exposure-related changes were observed in the nasal tissues, larynx, left lung lobe and tracheobronchial lymph nodes of animals of the main groups, histopathological examination of these tissues was extended to animals of the recovery groups.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
The lungs of all animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 mL saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 mL saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations. Samples of bronchoalveolar lavage fluid were discarded after analysis.
- Biochemical determinations in BAL fluid: The volume of the supernatant was determined. Total protein, phospholipids and lactate dehydrogenase (LDH) were determined.
- Cellular determinations in BAL fluid: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grünwald Giemsa. The differential cell counts were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
Since exposure-related changes were observed in animals of the main groups, investigation of biochemical and cellular BAL parameters was extended to animals of the recovery groups.

Statistics:

- Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. Day 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data pre-processing.
- Pre-treatment body weight, organ weight, hematology, clinical chemistry, and bronchoalveolar lavage data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method.
- Food consumption: Dunnett’s multiple comparison test.
- Incidences of histopathological changes: Fisher’s exact probability test.
- Arithmetic means and standard deviations are given in the tables of continuous and semicontinuous data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed in a few animals across the groups; these are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A male animal of the high-concentration recovery group was humanely sacrificed on day 73, because it was suffering from an othematoma (a hematoma in the external ear) which was not expected to heal and was probably causing substantial distress given the swelling and the restraint in the restraining tube required for nose-only exposure. This finding was not related to the exposure to the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related changes in body weight up to the highest concentration tested. A statistically significantly increased terminal body weight in females of the low- (but not of the mid- and high-) concentration group at the end of the exposure period was evidently a chance finding.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption were observed; food consumption was comparable across the groups throughout the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination did not reveal any exposure-related abnormalities.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Hematology Results Main groups and Hematology Results Recovery groups)
- Increased numbers of neutrophils in blood of animals of the mid- and high-concentration group. As a consequence, the total white blood cell count was increased in males of the high-concentration group, and the relative contribution of lymphocytes was decreased in females of the mid- and high-concentration and in males of the high-concentration group.
Since exposure-related changes were observed in animals of the main groups, examination of white blood cell parameters was extended to animals of the recovery groups. At the end of the 13-week recovery period, a statistically significant increase in the absolute and relative number of neutrophils was still observed for females of the mid- and high-concentration, and males of the high-concentration group. Consequently, total white cell numbers were increased in animals of the high-concentration (both sexes), and the percentage of lymphocytes was decreased in females of the mid- and high-concentration group. In addition, a slight increase in absolute eosinophil count was observed in females of the high-concentration recovery group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Analysis of clinical chemistry parameters did not reveal any treatment-related changes in animals exposed to the test material.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Relative Organ weights Main groups and Relative Organ Weights Recovery groups)
- Increased absolute and relative lung weight in animals of the mid- and high-concentration group. On average, relative lung weights were increased by 29% (males) to 41% (females) at the mid-concentration, and by 131% (females) to 135% (males) at the high concentration level.
- At the end of the recovery period, increased lung weights were still observed in animals of the mid- and high-concentration groups but the increase for the mid-concentration group was lower than at the end of the treatment period (+21% for females and + 26% for males) while it was more pronounced for the high-concentration group (+166% for females and + 180% for males.
- A statistically significantly decreased relative spleen weight in males of the mid-concentration recovery group was considered to be a chance finding, because a concentration-response relationship was absent. Absolute spleen weights were slightly lower than controls in males of the low- and high concentration group. Again, a concentration-response relationship was lacking; this finding (which was not observed in animals of the main groups, or in females of the recovery groups) was likely related to the slightly lower average body weight, and was not considered to be of any toxicological relevance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy of animals of the main groups, macroscopic examination revealed a concentration related increase in the incidence of tracheobronchial lymph node enlargement. Lymph node enlargement was found in 6/20 animals of the mid-concentration and 12/20 animals of the high concentration main groups. In addition,
12/20 high-concentration rats showed white spots on the lungs.
These macroscopic lesions were also observed in animals sacrificed at the end of the recovery period. Necropsy revealed white spots on the lungs of 10/20 animals of the mid-concentration and 19/19 animals (note that one male was humanely sacrificed before scheduled termination) of the high-concentration recovery group.
Tracheobronchial lymph node enlargement was found in 17/20 animals of the mid-concentration and 19/19 animals of the high concentration recovery group.
The remaining gross changes observed, represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Microscopic Examinations Main groups, Microscopic Examinations Recovery groups and Historical control Data)
Microscopic evaluation revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes of animals of the main groups

- The changes in the nose were predominantly present in the caudal parts and were characterized by a concentration-related increase of hyaline droplets in the olfactory epithelium at levels 4, 5 and 6. This finding was mainly observed in animals of the mid- and high-concentration groups and reached statistical significance only at nose level 5 for male animals of the high concentration group. Hyaline droplets graded as minimal were also observed in one animal of the low –concentration and control groups. Hyaline droplets graded as minimal were also observed in one animal of the low –concentration and control groups. At the end of the recovery period,hyaline droplets were still present, primarily in animals of the mid- and high-concentration, but also in single animals of the control and low concentration recovery groups.

- Changes in the larynx comprising squamous metaplasia of the lining epithelium, with minimal focal metaplasia were observed in1/20 animals of the mid-concentration, and minimal to mild focal metaplasia were noted in 8/20 animals of the high-concentration group. Exposure-related changes in the larynx were no longer observed at the end of the 13-week recovery period.

- In the lungs, a concentration-related increase of alveolar inflammation was observed, characterized by the presence of a mixed population of inflammatory cells (mononuclear cells, macrophages and/or granulocytes) at the end of the treatment period. Minimal focal and multifocal alveolar inflammations were found in 3/20 and 1/20 animals of the low-concentration respectively.Mild to moderate alveolar inflammation was found in all animals of the mid- and high-concentration groups; at the mid-concentration, multifocal lesions were observed, while at the high-concentration, the inflammation was found diffusely in the lungs.
In addition, minimal to mild perivascular mononuclear inflammation was observed in the lungs of 19/20 animals of the mid-concentration and 20/20 animals of the high concentration group. In the high-concentration group, granulomata (micro granulomas) were also found in 14/20 animals.
Furthermore, a concentration-related increase in the presence of intra-alveolar granular material was observed, with a minimal presence in the low-concentration (1/20 animals), mild to moderate multifocal presence in the mid-concentration (20/20 animals) and moderate to marked diffuse presence in the high-concentration group (20/20 animals).
At the end of the recovery period, minimal (multi)focal alveolar inflammation was found in 10/20 animals of the low concentration recovery group. At the mid-concentration, minimal to moderate multifocal alveolar inflammation (19/20 animals) and moderate diffuse alveolar inflammation (1/20 animals) was found. In the high-concentration group, moderate multifocal alveolar inflammation (1/19 animals) and mild to moderate diffuse alveolar inflammation (18/19 animals) was found. In addition, minimal to mild perivascular mononuclear inflammation was observed in the lungs of 15/20 animals of the mid-concentration and minimal to moderate perivascular mononuclear inflammation was observed in 17/19 animals of the high-concentration group. Granulomata were found in the lungs of 17/19 animals of the high-concentration, but also in 17/20 animals of the mid-concentration group. Intra-alveolar granular material was still present in animals of the mid- and high-concentration recovery group (graded as minimal to moderate at the mid-concentration, and mild to marked at the high-concentration level).

- Histopathological examination of the draining tracheobronchial lymph nodes revealed minimal to mild multifocal accumulation of macrophages in 15/20 animals of the mid-concentration and 15/20 animals of the high-concentration group. Although not statistically significant, mild to moderate lymphoid hyperplasia was also found in the mid- (2/20 animals) and the high concentration groups (3/20 animals). At the end of the recovery period, examination of tracheobronchial lymph nodes revealed multifocal accumulation of macrophages in 2/20 animals of the low-concentration (graded as minimal),19/20 animals of the mid-concentration (mild to moderate) and 19/19 animals of the high-concentration(mild to marked).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Bronchoalveolar Lavage Results Main groups and Bronchoalveolar Lavage Results Recovery groups)
Bronchoalveolar lavage (BAL)
Analysis of BAL parameters revealed the following differences between animals of the main groups exposed to the test material and unexposed controls:
- Increase in phospholipids, total protein content and lactate dehydrogenase activity (LDH) in BAL of males and females of the mid- and high-concentration group. Phospholipids were also slightly elevated in females of the low-concentration group.
- Increase in the absolute number of neutrophils and macrophages in BAL fluid of animals of the mid- and high-concentration group. Absolute neutrophil numbers were also slightly elevated in females of the low-concentration, and lymphocyte numbers were slightly increased in females of the mid- and high-concentration and males of the highconcentration group.
- As a result, animals of the mid- and high-concentration group showed statistically significantly increased total cell counts in BAL fluid, with a shift in the percentage distribution of white blood cells from ~100% macrophages (which is normally observed in healthy animals) towards an increased relative contribution of neutrophils and – to a lesser extent – lymphocytes.
It must be mentioned that the evaluation of differential cell numbers in animals of the highconcentration group was complicated by the presence of large amounts of debris in the cytospins. This debris was also found in BAL fluid of animals of the recovery groups, and prevented the reliable assessment of cell differentials of animals of the high-concentration recovery group.
In general, the above-described changes in BAL parameters in animals of the main groups, were still observed at the end of the 13-week recovery period:
- The levels of phospholipids, total protein and LDH were statistically significantly increased in animals of the mid- and high-concentration recovery group when compared to concurrent controls.
- The absolute numbers of neutrophils and lymphocytes were increased in animals of the mid-concentration group, and females also showed an increased number of macrophages (again, note that the high-concentration group could not be evaluated). Consequently, the percentage of neutrophils and lymphocytes was increased and the percentage of macrophages was decreased in animals of the mid-concentration group. Total cell numbers in BAL fluid were statistically significantly increased in females of the mid- and high-concentration, and males of the highconcentration group at the end of the recovery period.

Key result

Dose descriptor:

NOAEC

Remarks:

Local effects

Effect level:

3.06 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: changes in bronchoalveolar lavage markers

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

103.4 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no exposure-related systemic toxicity observed, all changes are related to the local toxicity

Key result

Critical effects observed:

no

Conclusions:

The results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 3.06 mg/m3.

Executive summary:

The toxicity of the test substance upon repeated exposure by inhalation was investigated in a 90-day sub-chronic study in Wistar rats according to OECD 413 and Good Laboratory Practices.

Four main groups of ten male and ten female rats each were exposed nose-only to target concentrations of 0 (control), 3, 20 or 100 mg/m3 for 6 hours/day, 5 days/week over a 90-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, recovery groups – also consisting of ten male and ten female animals each – were simultaneously exposed with animals of the main groups and were sacrificed after a 13-week recovery period following the last exposure.

Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobes together with the full respiratory tract and a large number of organs and tissues were examined microscopically.

The average Mass Median Aerodynamic Diameter of the particles in the test atmosphere was 2.38, 2.42 and 2.15 µm for the low-, mid- and high-concentration groups, respectively while the achieved aerosol concentrations for the corresponding groups were 3.06, 20.5 and 103.4 mg/m3. Chemical analysis of test atmosphere samples by UPLC-MS/MS indicated that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period.

Exposure-related mortality did not occur during the study. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities. Growth and food consumption were not adversely affected by the exposure and were comparable across the groups.

Analysis of haematology parameters revealed an increase in neutrophil numbers in blood of animals of the mid- and high- concentration group at the end of the exposure period, which was considered to be related to the inflammatory changes in the lower airways. Consequently, total white blood cells were increased in males of the high-concentration group, and the relative contribution of lymphocytes was decreased in females of the mid- and high concentration and in males of the high-concentration group. At the end of the recovery period, these changes in total and differential white blood cells were still found. Exposure to the test material did not result in any changes in clinical chemistry parameters.

Analysis of bronchoalveolar lavage (BAL) parameters revealed a concentration-related increase in total protein, phospholipids and lactate dehydrogenase activity, and increased numbers of (mainly) neutrophils and macrophages in BAL fluid of animals of the mid- and high concentration main groups. Lymphocyte numbers were also slightly increased in females of the mid- and high concentration and males of the high-concentration main groups. Consequently, the percentage distribution of white blood cells in BAL fluid shifted from ~100% macrophages – which is normally observed in healthy animals – towards an increased contribution of primarily neutrophils in animals of the mid- and high-concentration group. Comparable changes were generally still observed in animals sacrificed at the end of the recovery period.

Organ weight data showed an increase in absolute and relative weight of the lungs in animals of the mid- and high-concentration group at the end of the exposure period, and at the end of the recovery period. On average, relative lung weights were increased by 29% (males) to 41% (females) at the mid-concentration, and by 131% (females) to 135% (males) at the high-concentration level which was a indicative of a pulmonary overload.

Macroscopic examination at scheduled termination revealed white spots on the lungs of 12/20 animals of the high-concentration main group while at the end of the recovery period

all animals of the high-concentration recovery group and 10/20 animals of the mid-concentration recovery group showed white spots.

In addition, enlargement of the tracheobronchial lymph nodes was found in 6/20 and 12/20 animals of the mid- and high concentration main groups, and in 17/20 and 19/19 (note that one male was humanely sacrificed before scheduled necropsy) of the mid- and high-concentration recovery groups, respectively.

Microscopic examination revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes of animals of the mid- and high-concentration main groups.

The changes in the nose were characterized by an increased presence of hyaline droplets in the olfactory epithelium in the caudal parts of the nasal tissues. The incidence of this finding, observed in several animals at the end of the exposure period, was slightly higher at the end of the recovery period.

Histopathological changes in the larynx consisted of focal squamous metaplasia of the lining epithelium, observed in 1/20 and 8/20 animals of the mid- and high-concentration main groups, respectively. No exposure-related laryngeal abnormalities were found at the end of the recovery period.

The changes in the lungs were characterized by alveolar inflammation (containing a mixed population of mononuclear cells, macrophages and/or granulocytes) associated with the presence of intra-alveolar granular material in all animals of the mid- and high-concentration main and recovery groups. Perivascular mononuclear inflammation was observed in (almost) all mid- and high-concentration main and recovery animals. Granulomata (micro granulomas) were found in the lungs of 14/20 animals of the high-concentration main group, and in 17/20 and 17/19 of the mid- and high-concentration recovery groups, respectively. The microscopic findings in the lungs were consistent with those described in the literature following inhalation of inert particles and can be attributed to macrophage overload with inhaled test material and subsequent incomplete clearance leading to macrophage accumulation in the terminal air spaces.

Microscopic accumulations of macrophages were observed in the tracheobronchial lymph nodes of the majority of animals of the mid- and high-concentration main groups, accompanied by lymphoid hyperplasia in some animals. An increased incidence of these lesions was seen in animals sacrificed at the end of the recovery period. The changes in the local lymph nodes were considered to be a secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

In addition to the above-described local toxicity, the extensive histopathological examination of organs and tissues did not reveal any indications for systemic toxicity in response to the exposure to the test material. In contrast to the evident pathology observed in the mid- and high-concentration groups, very limited changes were found in the low-concentration group. Histopathological examination of the lungs revealed a minimal amount of intra-alveolar matter in the lungs of only one animal of the low-concentration main group. Minimal alveolar inflammatory foci were observed in four animals of the low-concentration main group and ten animals of the recovery group. While not observed in concurrent controls, minimal to mild (multi- ) focal alveolitis can be observed in control animals as common background pathology. Given the absence of any further tissue reaction, these minimal findings were considered to be below a level of toxicological concern.

In conclusion, the results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 3.06 mg/m3. Systemic toxicity was not observed up to concentrations of 103.4 mg/m3.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

103.4 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Subacute and suchronic inhalation toxicity studies available. The latest with 13 weeks of recovery is complete and sufficient to fulfill the REACh annex IX requirements

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 06 November 2012 to 14 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

This pilot study was performed in order to obtain preliminary information about the local and systemic toxicity of the substance when administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats. The results of the study are intended to be used for dose level selection for the 90-day inhalation study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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
- Age at study initiation: Young adult rats, 9 weeks old
- Weight at study initiation: 171 to 299 g (males: 268-299 g; females: 171-207g)
- Housing: group caging (5 animals/sex/cage)
- Bedding: Lignocel bedding for laboratory animals was available to animals during the study
- Diet (e.g. ad libitum): Ssniff SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance" ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22±3 °C
- Humidity: 30–70 %
- Air changes: 15-20 air exchanges/h
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light

Deviation:The temperature (22±3ºC) and the humidity (30–70%) values deviated from the required range during the acclimation period in the animal room. However, these deviations had no effect on the purpose and integrity of the study.

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1.97 - <= 2.73 µm

Geometric standard deviation (GSD):

2.9

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 1.97-2.73 µm with Geometric Standard Deviation (GSD) of 2.90-3.22. Due to the test item physical properties the Geometric Standard Deviation (GSD) of the test atmosphere was slightly above 3, but was kept in range of 3±10%.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus:TSE Rodent Exposure System (TSE Systems GmbH, Bad Homburg, Germany) which is a flow-past, nose-only exposure unit. This system consists of two, concentric anodised aluminium cylinders, the inner plenum and the outer chamber with 20 circularly arranged exposure ports.The equipment is supported by a computer control system incorporating pressure detectors, mass flow controllers as well as temperature, O2 and CO2 sensors.

- Method of holding animals in test chamber: animals were held in polycarbonate restraint tubes located around the chamber which allow only the animals’ nares to enter the exposure port.

- Source of air: compressed air

- Method of conditioning air: the compressed air was passed through a respiratory quality filter train and condensate separator prior to use.

- System of generating particulates: rotating brush powder disperser (Palas GmbH, Karlsruhe, Germany) located at the top of the exposure chamber

- Temperature, humidity, pressure in air chamber: 22±3 °C, 30–70 % and pressure in air chamber not reported

- Air flow rate: the flow of air through each port was at least 0.5 L/min.

- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 7-stage cascade impactor of Mercer sty le (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected at least once during each week of exposure for each concentration tested. Samples were also collected from a vacant animal exposure port (animals breathing zone). The collection substrates and the backup filter were weighed before and after sampling and the weight of test item, collected at each stage, was calculated by this difference. The total amount collected for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than < 0.550, 0.550, 0.960, 1.550, 2.105, 3.555, 6.655 and 10.550 µm was calculated. From this data, using the software supplied with the impactor (TSE Systems GmbH, Bad Homburg, Germany), the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated. In addition, the proportion (%) of aerosol less than 3 µm was determined.

- Treatment of exhaust air: After passing through the animal’s breathing zone, spent aerosol enters the outer cylinder from where it was exhausted through a suitable filter system.

TEST ATMOSPHERE
- Brief description of analytical method used: The actual 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, ref. no. 10370302). Sampling was normally performed shortly after chamber equilibration and then at regular intervals (approximately hourly intervals) during the exposure and samples were collected from a vacant animal exposure port (animals breathing zone). The difference in the pre- and post-sampling weights, divided by the volume of atmosphere sampled, was equal to the actual achieved test atmosphere concentration.

- Samples taken from breathing zone: yes, samples were collected from a vacant animal exposure port.

VEHICLE (if applicable)
- none

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The exposure concentrations were monitored intermittently by gravimetrical analysis of the test item deposited on a sampling filter. Sometimes, marked fluctuations in sample concentration occurred. These fluctuations can be explained by the short duration of the sampling, however the average concentration was equal to the target concentrations. The nominal concentration (mass of the test item dispersed into the exposure system in total air flow used for exposure) deviates significantly from mean achieved concentration due to loss of particles in pre-separation devices used for particle size optimization.
The mean achieved concentrations were 0.10; 0.50 and 2.02 mg/L and corresponded to 100; 100 and 101% of the target concentrations respectively.

Duration of treatment / exposure:

14 days

Frequency of treatment:

5 days/ week

Dose / conc.:

100 mg/m³ air (analytical)

Remarks:

Achieved concentration for low-concentration group

Dose / conc.:

500 mg/m³ air (analytical)

Remarks:

Achieved concentration for mid-concentration group

Dose / conc.:

2 020 mg/m³ air (analytical)

Remarks:

Achieved concentration for high-concentration group

No. of animals per sex per dose:

5

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The target concentration levels were 0.10, 0.50 and 2.0 mg/L of test item. They were set on the basis of available data and information from previous experimental work, including the results of an acute inhalation toxicity study (see section 7.2.2). In this 4–hour exposure study, no death occurred in group of six male and female rats exposed to a mean achieved atmosphere of 5.05 mg/l.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical observations were performed prior to exposure on the working days, and at least twice during the 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. During the weekend (no exposure) the clinical signs were recorded once per day only.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed clinical observations were made on all animals outside the home cage weekly, on Day 1, 7, 14.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded with a precision of 1 g at randomization, then on Day 1 and at least on Days 4, 8, 11, 14 and 15 (prior to necropsy, fasted before the scheduled euthanasia).

FOOD CONSUMPTION: Yes
- Food was recorded on Day 1 and at least on Days 4, 8, 11 and 14. The remaining, non-consumed food given was weighed with a precision of 1g for each cage. The mean individual daily food consumption was calculated per animal.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 15
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 15
- Animals fasted: Yes
- How many animals: all animals of all groups
- Parameters checked in table 7.5.2/2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross necropsy and macroscopic examination were performed on all animals. After exsanguination, the external appearance was examined, cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormalities were recorded with details of the location, colour, shape and size, as appropriate.

HISTOPATHOLOGY: Yes
Initial processing was limited to larynx, trachea, lungs, tracheobronchial lymph nodes, nasal cavity and any gross abnormalities for all animals of all groups.

Other examinations:

Yes
The following organs were weighed : Brain, heart, kidneys, liver, lungs, spleen, testes, thymus, adrenals and ovaries.

Statistics:

The heterogeneity of variance between groups were checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan’s Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data was not normally distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons was performed using Mann-Whitney U-test.

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

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, treatment-related

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There was no mortality observed during the study.
In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
• A slight increase in respiratory rate was observed in 4 out of 5 females on Day 1 and in 2 out of 5 males on Days 3-4,
• Slight and on a few occasions moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3 out of 5 animals,
• Slight noisy respiration turning to moderate in two occasions, was observed from Day 2 to Day 10 in 1 to 2 females,
• Hunched posture was noted from Day 4 to Day 12 in 1 to 3 females and on Days 10-11 in 1 male,
• Sneezing was noted for 2 females on Days 10-11.

In the animals exposed to the concentrations of 0.50 mg/L, no significant clinical signs were noted except for 1 male where initially slight subcutaneous mass in the left axillary was recorded from Day 5 which continuously grew until the end of the study.

In the animals exposed to the concentrations of 0.10 mg/L, slightly laboured respiration was noted in 1 female on day 5. No significant clinical signs were noted for the other animals in this group during the exposure period.

No clinical signs were recorded for the air control animals.


BODY WEIGHT AND WEIGHT GAIN
In the animals exposed to the concentrations of 2.02 mg/L,, a very slight mean bodyweight loss was recorded during the first days of treatment in both sexes. In comparison with controls, the decrease in mean bodyweight during days 1 to 4 was -2.2% and -0.43% in males and females respectively. In addition, females showed a statistically significant decrease in mean bodyweight gain during the overall period of treatment (-36% compared with controls).

No effects on bodyweight and bodyweight gain were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

FOOD CONSUMPTION
In the animals exposed to the concentrations of 2.02 mg/L, there was a significant decrease in food consumption in both males and females. The lower food consumption was in accordance with the effect on body weight.

No effects on food consumption were noted in the animals exposed to 0.50 or 0.10 mg/L when compared with controls.

HAEMATOLOGY
An increase in White Blood Count (WBC) was observed in both sexes at all doses but without dose-dependency. In parallel, increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed during the hematology analysis. Slightly decreased of Red Blood Count anda slight increase of Mean Cell Haemoglobin were observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered toxicologically significant (see table 7.5.2/1).

CLINICAL CHEMISTRY

There was no effect of treatment on clinical chemistry.

ORGAN WEIGHTS
The mean absolute and relative lungs weights, adjusted for both brain and terminal body weight, were significantly higher in all treated groups. They increased in a dose dependent manner up to 2 times in the 2.02 mg/L Group (see table 7.5.2/2).

GROSS PATHOLOGY
There was evidence of test item-related macroscopic changes in the lungs at the dose levels of 0.50 and 2.02 mg/L.
Enlargement/pale discoloration were observed in 5 out of 5 males and 5 out of 5 females in the 2.02 mg/L group. Pale foci were seen in 2 out of 5 females of the 0.50 mg/L group.

HISTOPATHOLOGY: NON-NEOPLASTIC
Test item-related findings were noted in the lungs and the tracheobronchial lymph nodes at all dose levels. There was clear relationship of these microscopic findings to the dose.
The alterations in the lungs were characterized by the presence of white/pink foreign material in the alveoli which was granular in consistency. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose.
The inflammatory alterations in the lungs ranged from minimal to moderate severity, with the presence of a mixed cell population (prominent feature of lymphocytes, macrophages and neutrophils) in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently to dose. Additionally, the formation of microgranulomas was shown by light microscopy in the alveoli of animals treated at 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes. The low severity (minimal) at0.1 mg/L, increased (mild) at 0.50 mg/L and was moderate at 2.02 mg/L (see table 7.5.2/3).

Key result

Dose descriptor:

LOAEC

Effect level:

100 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: At 100 mg/m3, slight increase in neutrophils and slight concomitant decrease in lymphocytes were observed during the Hematology analysis. These effects were associated with a slight inflammation process in the respiratory tract of the animals

Critical effects observed:

no

Table 7.5.2/1 Mean values in affected haematologic parameters

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
White Blood Count – WBC (K/mL)	2.776	3.244	3.664	3.314	NS
Red Blood Count – RBC (M/mL)	8.726	8.384	8.272	8.096*	DN
Neutrophils –NEU, Relative (%)	25.160	21.480	28.320	34.720**	DN
Lymphocytes – LYMPH, Relative (%)	69.620	74.080	66.780	59.040**	DN
Mean Cell Haemoglobin – MCH, pg	18.18	19.00**	19.12**	19.06**	DN
Females
White Blood Count – WBC (K/mL)	1.216	2.394*	2.130*	1.948*	U
Red Blood Count – RBC (M/mL)	7.932	8.108	7.986	8.034	NS
Neutrophils –NEU, Relative (%)	14.660	20.920*	20.420*	26.360**	DN
Lymphocytes – LYMPH, Relative (%)	80.460	73.920*	74.480*	67.900**	DN
Mean Cell Haemoglobin – MCH, pg	18.94	19.32	19.18	19.62	NS

NS = Not Significant

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.2/2 Mean values of lungs weights following the 14-day exposure period

GROUP	1 (Control)	2 (Low)	3 (Mid)	4 (High)	Remarks
Concentration (mg/L)	0	0.1	0.5	2.02
Males
Lungs Weight (g)Absolute	1.318	1.620**	2.092**	2.884**	U
Relative to Brain Weight (%)	69.52	83.78**	109.68**	145.41**	U
Relative to Body Weight (%)	0.44	0.54**	0.68**	0.99**	DN
Females
Lungs Weight (g)Absolute	1.014	1.286**	1.804**	2.256**	DN
Relative to Brain Weight (%)	55.01	73.04**	98.92**	124.25**	DN
Relative to Body Weight (%)	0.51	0.64**	0.88**	1.17**	DN

DN =Duncan's Multiple Range Test

U = Mann- Whitney U-Test

*= p<0.05

** = p<0.01

Table 7.5.3/3 Summary of histopatology data for lungs and tracheobronchial lymph nodes

Sex	male	male	male	male	female	female	female	female
Concentration (mg/l)	0	0.10	0.50	2.02	0	0.10	0.50	2.02
Number of Animals on Study	5	5	5	5	5	5	5	5
LUNGS;
Examined	(5)	(5)	(5)	(5)	(5)	(5)	(5)	(5)
WithinLimits	5	0	0	0	5	0	0	0
Foreign Material; alveolar; all lobes, Glanular, white/pink	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
minimal	0	4	1	0	0	5	0	0
mild	0	1	4	0	0	0	3	0
Foreign Material; alveolar; all lobes, Glanular, white/pink; correlated with necropsy.	(0)	(5)	(5)	(5)	(0)	(5)	(5)	(5)
mild	0	0	0	1	0	0	2	2
moderate	0	0	0	4	0	0	0	3
Inflammation, Bronchioloalveolar; mixed; all lobes	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
minimal	0	0	5	0	0	0	5	0
mild	0	0	0	4	0	0	0	1
moderate	0	0	0	1	0	0	0	1
Inflammation, Bronchioloalveolar; mixed; all lobes correlated with necropsy	(0)	(0)	(5)	(5)	(0)	(0)	(5)	(5)
mild	0	0	0	4	0	0	0	3
Microgranuloma; alveolar; multifocal	(0)	(0)	(0)	(3)	(0)	(0)	(0)	(3)
minimal	0	0	0	1	0	0	0	2
mild	0	0	0	2	0	0	0	1
Cell Infiltrate, Mixed Cellular; bronchiole; alveolus, all lobes	(0)	(5)	(0)	(0)	(0)	(5)	(0)	(0)
minimal	0	3	0	0	0	5	0	0
mild	0	2	0	0	0	0	0	0
LYMPH NODE, LUNG-ASSOCIATED;
Examined	(5)	(5)	(5)	(5)	(4)	(5)	(5)	(5)
WithinLimits	5	0	0	0	4	2	0	0
Not Examined: NOT PRESENT	0	0	0	0	1	0	0	0
Accumulation, Foamy Macrophage; medulla; paracortex	(0)	(5)	(5)	(5)	(0)	(3)	(5)	(5)
minimal	0	2	1	0	0	3	2	0
mild	0	3	4	0	0	0	3	1
moderate	0	0	0	5	0	0	0	4

Conclusions:

Exposure to the test substance at gravimetrically determined dose levels of 100, 500 and 2020 mg/m3 air resulted in findings in the lungs and tracheobronchial lymph nodes. In the lungs, they consisted of inflammatory reactions ranging from minimal to moderate severity, with the presence of a mixed cell population. Also, foreign material was seen in the alveoli. The incidence and severity of the inflammatory responses and the presence of foreign alveolar material were generally increased consistently to dose. A similar pattern of dose relationship was recorded for the accumulation of macrophages in the tracheobronchial lymph nodes. The low severity (minimal) in Low dose, increased (mild) in Mid dose and was moderate in High dose animals.

Executive summary:

In a pilot study performed in compliance with Good Laboratory Practice, the test substance was administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats.

The test item was administered as a dry aerosol. The animals were exposed on 10 occasions during a 14 day period. Three groups, each comprising five male and five female rats received the test substance at achieved exposure levels of of 100, 500 and 2020 mg/m3. A similarly constituted Control group received air with the same operating conditions as the other groups.

The mortality observations were performed twice daily. The general clinical observations were recorded five times for the exposed animals on the days of exposure. Detailed clinical examination was performed on Days 1, 7 and 14 prior to the 6 hour exposure. Body weight and food consumption were measured on Days 1, 4, 8, 11 and 14. Blood samples were collected for clinical pathology at necropsy. Gross macroscopic examination was performed at necropsy one day after the last treatment. Selected organs were weighed. Histopathology investigation was performed on selected tissues.

The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3 µm) for a repeat dose inhalation study. 

There was no mortality during the study. Clinical signs related to treatment were noted only in animals given 2020 mg/m3. All but one female and only 2 males had a transient slight increase in respiratory rate. In females, slight and in few occasions moderate laboured and/ or noisy respiration was observed throughout the study. In males, slightly laboured respiration was noted only on day 2 in 3 animals. Hunched posture was observed from day 4 to day 10 in 1 to 3 females and on days 10-11 in 1 male. In addition, sneezing was noted for two females on Day 10 and 11. 

Changes in bodyweight and bodyweight gain were noted only in animals that received 2020 mg/m3. Temporary mean bodyweight loss was observed at the beginning of the study in both sexes on (Days 1-4) ( -2.2% and -0.43% in males and females respectively). This bodyweight loss correlated with the significant decrease in food consumption noted during the same treatment-period. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Following 2 weeks of treatment, an increase in White Blood Count was observed in both sexes at all doses but without dose-dependency. In addition, statistically significant increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed in males given 2020 mg/m3 and in all treated females. These changes in haematology might be contributed to changes in the lungs. A slight decrease in Red Blood Count and a slight increase of Mean Cell Haemoglobin were also observed in males. However the changes were slight in magnitude, and were within the physiological range. Therefore these changes were not considered to be toxicologically significant. There was no effect of treatment noted during the evaluation of the clinical chemistry parameters.

Macroscopic examination revealed enlargement and/or pale discoloration of the lungs in animals given 500 or 2020 mg/m3. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels. There was a clear relationship between these microscopic findings and the dose. The alterations in the lungs were characterized by the presence of white/pink foreign material in the alveoli. The presence of foreign alveolar material was generally minimal at low dose and moderate at high dose. The inflammatory reactions in the lungs ranged from minimal to moderate severity, with the presence of a mixed cell population in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently with dose. The formation of microgranulomas was observed microscopically in the alveoli of animals treated at a dose level of 2020 mg/m3. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in the Low dose group, increased (mild) in the Mid dose and moderate in the High dose group. The mean absolute and relative lung weights adjusted for both brain and terminal body weight were significantly higher in males and females at dose levels of 100, 500 and 2020 mg/m3 compared to the controls.

Based on these findings, it was anticipated that a target maximum exposure level of 100 mg/m3 would be tolerated for a 90 day exposure in the same species.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 Feb 2017 to 4 Aug 2017

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)

Version / remarks:

7 September 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

(Crl:WI(Han))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: ±20% of the mean weight for each sex which is 231 gr for males and 169 gr for females
- Housing: Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Envirodri,Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. Animals were housed in groups of five of the same sex. Upon reaching a size at which five/cage housing was no longer allowed, male animals were housed two or three per cage. During exposure, the animals were kept individually in the exposure unit
- Diet: cereal-based (closed formula) rodent diet (VRF1 (FG)) from a commercial supplier (SDSSpecial Diets Services, Witham, England) ad libitum.
- Water: Tap-water suitable for human consumption ad libitum
- Acclimation period: 13-16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45-65
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.15 - <= 2.42 µm

Geometric standard deviation (GSD):

1.82

Remarks on MMAD:

- The average Mass Median Aerodynamic Diameter (MMAD) of the particles in the test atmosphere was 2.38 (with a geometric standard deviation of the size distribution of 1.74), 2.42 (gsd of 1.82) and 2.15 µm (gsd of 1.84) for the low-, mid- and high-concentration groups, respectively.
- Particle size distribution measurements was carried out using a 10-stage cascade impactor at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: inhalation chambers consisting of a cylindrical aluminium column, surrounded by a transparent cylinder (a modification of the chamber made by ADG Developments Ltd., Codicote, Hitchin, Herts, SG4 8UB, United Kingdom).
- Method of holding animals in test chamber: animals were exposed to the test atmosphere in nose-only exposure units, in an illuminated lab room different than the room where the animals were housed. The animals were secured in plastic animal holders (Battelle), positioned radially through the outer cylinder around the central column. The remaining ports were closed. Only the nose of the rats protruded into the interior of the column.
- Source and rate of air: The average total air flows (± standard deviation) were 48.3 (± 0.3), 46.1 (± 0.1), 48.1 (± 2.0) and 66.6 (± 0.0) L/min for the control, low-, mid- and high-concentration groups, respectively
- - System of generating particulates/aerosols: The test atmosphere was generated using a turntable dust feeder (Reist and Taylor, 2000) and an eductor (Fox Valve Development Corp., Dover, NJ, USA; Cheng et al., 1989). The test material was aerosolized in the eductor, which was supplied with a flow (controlled using a reducing valve) of humidified compressed air. The resulting aerosol was led through a glass cyclone, which was used to remove the largest particles from the aerosol, and was subsequently introduced at the top inlet of the exposure chamber. The eductors were calibrated by measuring the total air flow at a range of driving air pressures encompassing the driving pressures used during the study. The driving air pressure was used to monitor the flow. A bypass stream of humidified compressed air (measured by mass view meter; Bronkhorst Hi Tec, Ruurlo, The Netherlands) was added at the top of the exposure chamber to ensure a relative humidity above 30% (for groups 2 and 3 only; this bypass flow of humidified air was not necessary for group 4). The resulting test atmosphere was directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted.
- Temperature, humidity, pressure in air chamber: The average temperature (± standard deviation) was 22.5 (± 0.4), 22.9 (± 0.4), 23.0 (± 0.4) and 23.4 (± 0.4)°C for the control, low-, mid- and high-concentration groups. The average relative humidity during exposure was 42.6 (± 1.4), 41.6 (± 2.8), 41.4 (± 2.1) and 40.2 (± 1.2) % for the control, low-, mid- and high-concentration groups, respectively.
- Air flow rate: The average total air flows (± standard deviation) were 48.3 (± 0.3), 46.1 (± 0.1), 48.1 (± 2.0) and 66.6 (± 0.0) L/min for the control, low-, mid- and high-concentration groups
- Method of particle size determination: using a 10-stage cascade impactor (2110k cascade impactor, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition.


TEST ATMOSPHERE
- Samples taken from breathing zone: test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) samples of test atmosphere through fibre glass filters (Sartorius 13400-47, Ø 47 mm) at a flow of 9.2 (group 2) or 4.6 (groups 3 and 4) Ln/min
- The chamber air flow was recorded at least hourly during exposure by recording the readings of the mass view meters and driving air pressure of the eductors.
- Temperature and the relative humidity of the test atmospheres were measured continuously during exposure, and recorded on a PC every minute using a CAN transmitter with temperature and relative humidity probes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Gravimetric analysis
The actual concentration of the test material in the atmosphere was measured at least three times during exposure on each exposure day by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) samples of test atmosphere through fibre glass filters (Sartorius 13400-47, Ø 47 mm) at a flow of 9.2 (group 2) or 4.6 (groups 3 and 4) Ln/min. Samples with a volume of 1002.4, 151.8 or 32.2 Ln were taken for group 2, 3 and 4, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and then weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter by the volume of the sample taken.

Chemical analysis
The content of the test material captured on the gravimetric filters was determined five times during the study by chemical analysis. Samples of the low-, mid- and high-concentrati n test atmosphere (and one blank) obtained during exposure of the animals in week 1, 4, 7, 10 and 13 were analyzed. The content of the test item on the filters was determined using ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS), using the following three main test substance constituents as markers to check whether the composition of the test material in the test atmospheres was similar to the original material and stable across the exposure period.

Duration of treatment / exposure:

13 weeks (ie 65 exposure days in total) followed by a 13- week recovery period.

Frequency of treatment:

6 hrs/day, 5 days / week.

Dose / conc.:

3.06 mg/m³ air (analytical)

Remarks:

Low concentration

Dose / conc.:

20.5 mg/m³ air (analytical)

Remarks:

Mid concentration

Dose / conc.:

103.4 mg/m³ air (analytical)

Remarks:

High concentration

No. of animals per sex per dose:

10 in both the main and recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The target concentrations were selected on the basis of the results of the two-week repeated inhalation toxicity study conducted in rats at exposure levels 0, 0.10, 0.50 and 2.02 mg/L (Nagy, 2013b). No animals died during the treatment period. At 2.02 mg/L, animals showed an increase in respiratory rate, laboured and/or noisy respiration. Reduced food consumption as well as reduced body weight gain and body weight loss were also observed. At 0.10 and 0.50 mg/L, there were no treatment-related clinical sings and no changes in body weight or food consumption. Treatment-related histopathological findings in the lungs and tracheobronchial lymph nodes were evident at all dose levels, however, the severity of the findings for animals that received 0.50 or 2.02 mg/L precluded these levels being selected for this study as it was unlikely that these concentrations would be tolerated for 13 weeks. As the findings in the lung were of lower severity at 0.10 mg/L, it was anticipated that a target exposure level of 100 mg/m3 will be tolerated for 13 weeks. To explore any possible concentration-response relationship, the target exposure levels for the mid- and low-concentration groups were selected at 21 and 3 mg/m3, respectively.
- Species / strain selection: The Wistar strain will be used because it is routinely used at the test facility for this type of studies.

Positive control:

no

Observations and examinations performed and frequency:

CLINICAL SIGNS
Animals were observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. Clinical signs for respiration, behavior, head, perineum, skin/fur, eyes, nose, mouth, ears, extremities (legs), tail, abdomen, testes, penis, urethra, urine, faeces, injection site and general signs were observed. The animals were also observed about halfway through the 6-hour exposure period, in particular to monitor any breathing abnormalities and restlessness; observation of other abnormalities was hindered due to the animals’ stay in restraining tubes. All animals were thoroughly checked again in the afternoon. All abnormalities, signs of ill health, reactions to treatment and mortality were recorded.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic observations were made prior to the start of exposure in all animals (on day -8 to -6) and towards the end of the exposure period in the animals of the control and high concentration groups (on days 86 to 88). Eye examinations were carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate. Since no exposure-related ocular changes were observed, eye examinations were not extended to the animals of the intermediate concentration groups at the end of the exposure period, or to animals of the recovery groups.

BODY WEIGHTS
The body weight of each animal was recorded once before the start of the exposure period: on day -4 or -5 for males, and on day -6 or -7 for females. These weights were used for animal allocation. During the exposure period, the animals were weighed just before exposure on the first day (day 0), and twice a week thereafter. Since no exposure-related effects were seen during the first 4 weeks of the study, body weight measurements were thereafter continued on a once a week basis for the remainder of the study duration. The animals were also weighed on the day before overnight fasting prior to necropsy, and on their scheduled sacrifice date in order to calculate the correct organ to body weight ratios.

FOOD CONSUMPTION
Food consumption of the animals was measured per cage by weighing the feeders. The results were expressed in g per animal per day. Food consumption was measured from day 0 over successive periods of 7 days, until the last week before sacrifice (i.e. including the recovery period) in which food consumption was measured over a 6-day period.

HAEMATOLOGY
Haematology was conducted at the end of the treatment period on all surviving animals of the main groups. Blood samples were taken from the abdominal aorta of overnight fasted rats (water was freely available) whilst under pentobarbital anesthesia at sacrifice. EDTA was used as anticoagulant, except for PT where citrate was used. The samples were discarded after analysis. In each sample the following determinations were carried out:
- haemoglobin (Hb)
- packed cell volume (PCV)
- red blood cell count (RBC)
- reticulocytes
- total white blood cell count (WBC)
- differential white blood cell count (lymphocytes, neutrophils, eosinophils, basophils and monocytes)
- prothrombin time (PT)
- thrombocyte count (platelet count)
The following parameters were calculated:
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin (MCH)
- mean corpuscular hemoglobin concentration (MCHC)
Since (possible) exposure-related changes were observed in animals of the main groups, investigation of total and differential white blood cell counts was extended to animals of the recovery groups.

CLINICAL CHEMISTRY
Clinical chemistry was conducted at the end of the treatment period on all surviving rats after overnight fasting, at the same time blood samples for hematology were collected. The blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. After analysis, remaining plasma was stored frozen (<-18°C) to enable reanalysis if necessary and then discarded. The following measurements were made in the plasma:
- alkaline phosphatase activity (ALP)
- bilirubin total
- aspartate aminotransferase activity (ASAT)
- cholesterol
- alanine aminotransferase activity (ALAT)
- triglycerides
- gamma glutamyl transferase activity (GGT)
- phospholipids
- total protein
- calcium (Ca)
- albumin
- sodium (Na)
- ratio albumin to globulin
- potassium (K)
- urea
- chloride (Cl)
- creatinine
- inorganic phosphate
- fasting glucose
Since no exposure-related changes were observed in animals of the main groups, investigation of clinical chemistry parameters was not extended to the animals of the recovery groups.

Sacrifice and pathology:

SACRIFICE AND MACROSCOPIC EXAMINATION
Surviving animals of the main groups were sacrificed at the end of the exposure period (day 91) in such a sequence that the average time of sacrifice was approximately the same for each group. Similarly, animals of the recovery groups were sacrificed at the end of the 3-month recovery period (day 175). The animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anesthesia (intraperitoneal injection of sodium pentobarbital) and then examined grossly for pathological changes. A thorough necropsy was also performed on the animal that was killed in moribund.
At scheduled sacrifice, the following organs of all surviving animals were weighed (paired organs together) as soon as possible after dissection to avoid drying. Relative organ weights (g/kg bw) were calculated from the absolute organ weight and the terminal body weight:
- adrenals
- brain
- heart
- kidneys
- liver
- lungs
- ovaries
- spleen
- testes
- thymus
- uterus

HISTOPATHOLOGY
For histopathological examination, samples of the following tissues and organs of all animals (main and recovery groups) were preserved in a 10% solution of Formalin in a neutral aqueous phosphate buffer (final formaldehyde concentration 4 per cent). The left lung lobes (after weighing) were infused with the fixative under ca. 15 cm water pressure to ensure fixation. The carcass containing any remaining tissues was retained in the fixative until completion of the histopathological examination and then discarded.
- adrenals
- aorta
- axillary lymph nodes
- brain (Three levels were examined microscopically (brain stem, cerebrum, cerebellum)
- cecum
- colon
- epididymides
- eyes (with optic nerve)
- exorbital lachrymal glands
- femur with joint
- heart
- kidneys
- liver
- lungs/trachea /larynx (The left lung lobe was examined microscopically at three levels, Three levels were examined microscopically (including a longitudinal section through the carina of the bifurcation) and Three levels (one including the base of the epiglottis) were examined microscopically)
- mammary glands (females)
- cervical lymph nodes
- nasopharyngeal tissue (with teeth) Six levels were examined microscopically (one including the nasopharyngeal duct and the draining lymphatic tissue.
- nerve peripheral (sciatic nerve)
- esophagus
- olfactory bulb
- ovaries
- pancreas
- parathyroids
- pharynx
- parotid salivary glands
- pituitary gland
- prostate
- rectum
- seminal vesicles with coagulating
- glands
- skeletal muscle (thigh)
- skin (flank)
- small intestines (duodenum, ileum and jejunum)
- spinal cord (Retained in vertebral column, at least three levels were examined microscopically (cervical, mid-thoracic and lumbar).
- spleen
- sternum with bone marrow
- stomach (Non-glandular and glandular parts were examined microscopically)
- sublingual salivary glands
- submaxillary salivary glands
- testes
- thymus
- thyroid
- tongue
- tracheobronchial (mediastinal)
- lymph nodes
- ureter
- urethra
- urinary bladder
- uterus (with cervix)
- all gross lesions

Slide preparation: Tissues to be examined were embedded in paraffin wax, sectioned and stained with hematoxylin and eosin. Unless required for histopathological examination, the tissues of the animals of the low- and mid-concentration main groups (groups 2 and 3) and the recovery groups were not processed.
Bone marrow: Bone marrow smears were prepared of all surviving animals of the main and recovery groups for possible future examination.
Histopathological examination: All preserved tissues of all animals of the control and high-concentration main groups and of the animal which was sacrificed in moribund condition, were examined histopathologically (by light microscopy). In addition, the respiratory tract (nasopharyngeal tissues, larynx, trachea and left lung lobe) and tracheobronchial lymph nodes of animals of the intermediate concentration groups of the main study were examined histopathologically. Furthermore, all gross lesions observed in rats of the intermediate concentration groups and animals of the recovery groups were examined microscopically. Histopathology will be subjected to a peer review system. Since exposure-related changes were observed in the nasal tissues, larynx, left lung lobe and tracheobronchial lymph nodes of animals of the main groups, histopathological examination of these tissues was extended to animals of the recovery groups.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
The lungs of all animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 mL saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 mL saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations. Samples of bronchoalveolar lavage fluid were discarded after analysis.
- Biochemical determinations in BAL fluid: The volume of the supernatant was determined. Total protein, phospholipids and lactate dehydrogenase (LDH) were determined.
- Cellular determinations in BAL fluid: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grünwald Giemsa. The differential cell counts were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
Since exposure-related changes were observed in animals of the main groups, investigation of biochemical and cellular BAL parameters was extended to animals of the recovery groups.

Statistics:

- Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with automatic data transformation. Day 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data pre-processing.
- Pre-treatment body weight, organ weight, hematology, clinical chemistry, and bronchoalveolar lavage data: ‘Generalized Anova/Ancova Test’ with automatic data transformation method.
- Food consumption: Dunnett’s multiple comparison test.
- Incidences of histopathological changes: Fisher’s exact probability test.
- Arithmetic means and standard deviations are given in the tables of continuous and semicontinuous data.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical abnormalities were observed. The few signs noted were considered unrelated to the exposure to the test material. Abnormalities of the skin or fur (sparsely haired areas, encrustations, skin wounds) were observed in a few animals across the groups; these are common findings, possibly caused by slight movement of the animals in the restraining tubes during exposure, resulting in slight irritation of the skin.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A male animal of the high-concentration recovery group was humanely sacrificed on day 73, because it was suffering from an othematoma (a hematoma in the external ear) which was not expected to heal and was probably causing substantial distress given the swelling and the restraint in the restraining tube required for nose-only exposure. This finding was not related to the exposure to the test material.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

There were no exposure-related changes in body weight up to the highest concentration tested. A statistically significantly increased terminal body weight in females of the low- (but not of the mid- and high-) concentration group at the end of the exposure period was evidently a chance finding.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No treatment-related changes in food consumption were observed; food consumption was comparable across the groups throughout the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination did not reveal any exposure-related abnormalities.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Hematology Results Main groups and Hematology Results Recovery groups)
- Increased numbers of neutrophils in blood of animals of the mid- and high-concentration group. As a consequence, the total white blood cell count was increased in males of the high-concentration group, and the relative contribution of lymphocytes was decreased in females of the mid- and high-concentration and in males of the high-concentration group.
Since exposure-related changes were observed in animals of the main groups, examination of white blood cell parameters was extended to animals of the recovery groups. At the end of the 13-week recovery period, a statistically significant increase in the absolute and relative number of neutrophils was still observed for females of the mid- and high-concentration, and males of the high-concentration group. Consequently, total white cell numbers were increased in animals of the high-concentration (both sexes), and the percentage of lymphocytes was decreased in females of the mid- and high-concentration group. In addition, a slight increase in absolute eosinophil count was observed in females of the high-concentration recovery group.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Analysis of clinical chemistry parameters did not reveal any treatment-related changes in animals exposed to the test material.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Relative Organ weights Main groups and Relative Organ Weights Recovery groups)
- Increased absolute and relative lung weight in animals of the mid- and high-concentration group. On average, relative lung weights were increased by 29% (males) to 41% (females) at the mid-concentration, and by 131% (females) to 135% (males) at the high concentration level.
- At the end of the recovery period, increased lung weights were still observed in animals of the mid- and high-concentration groups but the increase for the mid-concentration group was lower than at the end of the treatment period (+21% for females and + 26% for males) while it was more pronounced for the high-concentration group (+166% for females and + 180% for males.
- A statistically significantly decreased relative spleen weight in males of the mid-concentration recovery group was considered to be a chance finding, because a concentration-response relationship was absent. Absolute spleen weights were slightly lower than controls in males of the low- and high concentration group. Again, a concentration-response relationship was lacking; this finding (which was not observed in animals of the main groups, or in females of the recovery groups) was likely related to the slightly lower average body weight, and was not considered to be of any toxicological relevance.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At necropsy of animals of the main groups, macroscopic examination revealed a concentration related increase in the incidence of tracheobronchial lymph node enlargement. Lymph node enlargement was found in 6/20 animals of the mid-concentration and 12/20 animals of the high concentration main groups. In addition,
12/20 high-concentration rats showed white spots on the lungs.
These macroscopic lesions were also observed in animals sacrificed at the end of the recovery period. Necropsy revealed white spots on the lungs of 10/20 animals of the mid-concentration and 19/19 animals (note that one male was humanely sacrificed before scheduled termination) of the high-concentration recovery group.
Tracheobronchial lymph node enlargement was found in 17/20 animals of the mid-concentration and 19/19 animals of the high concentration recovery group.
The remaining gross changes observed, represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Microscopic Examinations Main groups, Microscopic Examinations Recovery groups and Historical control Data)
Microscopic evaluation revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes of animals of the main groups

- The changes in the nose were predominantly present in the caudal parts and were characterized by a concentration-related increase of hyaline droplets in the olfactory epithelium at levels 4, 5 and 6. This finding was mainly observed in animals of the mid- and high-concentration groups and reached statistical significance only at nose level 5 for male animals of the high concentration group. Hyaline droplets graded as minimal were also observed in one animal of the low –concentration and control groups. Hyaline droplets graded as minimal were also observed in one animal of the low –concentration and control groups. At the end of the recovery period,hyaline droplets were still present, primarily in animals of the mid- and high-concentration, but also in single animals of the control and low concentration recovery groups.

- Changes in the larynx comprising squamous metaplasia of the lining epithelium, with minimal focal metaplasia were observed in1/20 animals of the mid-concentration, and minimal to mild focal metaplasia were noted in 8/20 animals of the high-concentration group. Exposure-related changes in the larynx were no longer observed at the end of the 13-week recovery period.

- In the lungs, a concentration-related increase of alveolar inflammation was observed, characterized by the presence of a mixed population of inflammatory cells (mononuclear cells, macrophages and/or granulocytes) at the end of the treatment period. Minimal focal and multifocal alveolar inflammations were found in 3/20 and 1/20 animals of the low-concentration respectively.Mild to moderate alveolar inflammation was found in all animals of the mid- and high-concentration groups; at the mid-concentration, multifocal lesions were observed, while at the high-concentration, the inflammation was found diffusely in the lungs.
In addition, minimal to mild perivascular mononuclear inflammation was observed in the lungs of 19/20 animals of the mid-concentration and 20/20 animals of the high concentration group. In the high-concentration group, granulomata (micro granulomas) were also found in 14/20 animals.
Furthermore, a concentration-related increase in the presence of intra-alveolar granular material was observed, with a minimal presence in the low-concentration (1/20 animals), mild to moderate multifocal presence in the mid-concentration (20/20 animals) and moderate to marked diffuse presence in the high-concentration group (20/20 animals).
At the end of the recovery period, minimal (multi)focal alveolar inflammation was found in 10/20 animals of the low concentration recovery group. At the mid-concentration, minimal to moderate multifocal alveolar inflammation (19/20 animals) and moderate diffuse alveolar inflammation (1/20 animals) was found. In the high-concentration group, moderate multifocal alveolar inflammation (1/19 animals) and mild to moderate diffuse alveolar inflammation (18/19 animals) was found. In addition, minimal to mild perivascular mononuclear inflammation was observed in the lungs of 15/20 animals of the mid-concentration and minimal to moderate perivascular mononuclear inflammation was observed in 17/19 animals of the high-concentration group. Granulomata were found in the lungs of 17/19 animals of the high-concentration, but also in 17/20 animals of the mid-concentration group. Intra-alveolar granular material was still present in animals of the mid- and high-concentration recovery group (graded as minimal to moderate at the mid-concentration, and mild to marked at the high-concentration level).

- Histopathological examination of the draining tracheobronchial lymph nodes revealed minimal to mild multifocal accumulation of macrophages in 15/20 animals of the mid-concentration and 15/20 animals of the high-concentration group. Although not statistically significant, mild to moderate lymphoid hyperplasia was also found in the mid- (2/20 animals) and the high concentration groups (3/20 animals). At the end of the recovery period, examination of tracheobronchial lymph nodes revealed multifocal accumulation of macrophages in 2/20 animals of the low-concentration (graded as minimal),19/20 animals of the mid-concentration (mild to moderate) and 19/19 animals of the high-concentration(mild to marked).

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

See detailed results in attached background material (Bronchoalveolar Lavage Results Main groups and Bronchoalveolar Lavage Results Recovery groups)
Bronchoalveolar lavage (BAL)
Analysis of BAL parameters revealed the following differences between animals of the main groups exposed to the test material and unexposed controls:
- Increase in phospholipids, total protein content and lactate dehydrogenase activity (LDH) in BAL of males and females of the mid- and high-concentration group. Phospholipids were also slightly elevated in females of the low-concentration group.
- Increase in the absolute number of neutrophils and macrophages in BAL fluid of animals of the mid- and high-concentration group. Absolute neutrophil numbers were also slightly elevated in females of the low-concentration, and lymphocyte numbers were slightly increased in females of the mid- and high-concentration and males of the highconcentration group.
- As a result, animals of the mid- and high-concentration group showed statistically significantly increased total cell counts in BAL fluid, with a shift in the percentage distribution of white blood cells from ~100% macrophages (which is normally observed in healthy animals) towards an increased relative contribution of neutrophils and – to a lesser extent – lymphocytes.
It must be mentioned that the evaluation of differential cell numbers in animals of the highconcentration group was complicated by the presence of large amounts of debris in the cytospins. This debris was also found in BAL fluid of animals of the recovery groups, and prevented the reliable assessment of cell differentials of animals of the high-concentration recovery group.
In general, the above-described changes in BAL parameters in animals of the main groups, were still observed at the end of the 13-week recovery period:
- The levels of phospholipids, total protein and LDH were statistically significantly increased in animals of the mid- and high-concentration recovery group when compared to concurrent controls.
- The absolute numbers of neutrophils and lymphocytes were increased in animals of the mid-concentration group, and females also showed an increased number of macrophages (again, note that the high-concentration group could not be evaluated). Consequently, the percentage of neutrophils and lymphocytes was increased and the percentage of macrophages was decreased in animals of the mid-concentration group. Total cell numbers in BAL fluid were statistically significantly increased in females of the mid- and high-concentration, and males of the highconcentration group at the end of the recovery period.

Key result

Dose descriptor:

NOAEC

Remarks:

Local effects

Effect level:

3.06 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: changes in bronchoalveolar lavage markers

Key result

Dose descriptor:

NOAEC

Remarks:

Systemic toxicity

Effect level:

103.4 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no exposure-related systemic toxicity observed, all changes are related to the local toxicity

Key result

Critical effects observed:

no

Conclusions:

The results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 3.06 mg/m3.

Executive summary:

The toxicity of the test substance upon repeated exposure by inhalation was investigated in a 90-day sub-chronic study in Wistar rats according to OECD 413 and Good Laboratory Practices.

Four main groups of ten male and ten female rats each were exposed nose-only to target concentrations of 0 (control), 3, 20 or 100 mg/m3 for 6 hours/day, 5 days/week over a 90-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, recovery groups – also consisting of ten male and ten female animals each – were simultaneously exposed with animals of the main groups and were sacrificed after a 13-week recovery period following the last exposure.

Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobes together with the full respiratory tract and a large number of organs and tissues were examined microscopically.

The average Mass Median Aerodynamic Diameter of the particles in the test atmosphere was 2.38, 2.42 and 2.15 µm for the low-, mid- and high-concentration groups, respectively while the achieved aerosol concentrations for the corresponding groups were 3.06, 20.5 and 103.4 mg/m3. Chemical analysis of test atmosphere samples by UPLC-MS/MS indicated that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period.

Exposure-related mortality did not occur during the study. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities. Growth and food consumption were not adversely affected by the exposure and were comparable across the groups.

Analysis of haematology parameters revealed an increase in neutrophil numbers in blood of animals of the mid- and high- concentration group at the end of the exposure period, which was considered to be related to the inflammatory changes in the lower airways. Consequently, total white blood cells were increased in males of the high-concentration group, and the relative contribution of lymphocytes was decreased in females of the mid- and high concentration and in males of the high-concentration group. At the end of the recovery period, these changes in total and differential white blood cells were still found. Exposure to the test material did not result in any changes in clinical chemistry parameters.

Analysis of bronchoalveolar lavage (BAL) parameters revealed a concentration-related increase in total protein, phospholipids and lactate dehydrogenase activity, and increased numbers of (mainly) neutrophils and macrophages in BAL fluid of animals of the mid- and high concentration main groups. Lymphocyte numbers were also slightly increased in females of the mid- and high concentration and males of the high-concentration main groups. Consequently, the percentage distribution of white blood cells in BAL fluid shifted from ~100% macrophages – which is normally observed in healthy animals – towards an increased contribution of primarily neutrophils in animals of the mid- and high-concentration group. Comparable changes were generally still observed in animals sacrificed at the end of the recovery period.

Organ weight data showed an increase in absolute and relative weight of the lungs in animals of the mid- and high-concentration group at the end of the exposure period, and at the end of the recovery period. On average, relative lung weights were increased by 29% (males) to 41% (females) at the mid-concentration, and by 131% (females) to 135% (males) at the high-concentration level which was a indicative of a pulmonary overload.

Macroscopic examination at scheduled termination revealed white spots on the lungs of 12/20 animals of the high-concentration main group while at the end of the recovery period

all animals of the high-concentration recovery group and 10/20 animals of the mid-concentration recovery group showed white spots.

In addition, enlargement of the tracheobronchial lymph nodes was found in 6/20 and 12/20 animals of the mid- and high concentration main groups, and in 17/20 and 19/19 (note that one male was humanely sacrificed before scheduled necropsy) of the mid- and high-concentration recovery groups, respectively.

Microscopic examination revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes of animals of the mid- and high-concentration main groups.

The changes in the nose were characterized by an increased presence of hyaline droplets in the olfactory epithelium in the caudal parts of the nasal tissues. The incidence of this finding, observed in several animals at the end of the exposure period, was slightly higher at the end of the recovery period.

Histopathological changes in the larynx consisted of focal squamous metaplasia of the lining epithelium, observed in 1/20 and 8/20 animals of the mid- and high-concentration main groups, respectively. No exposure-related laryngeal abnormalities were found at the end of the recovery period.

The changes in the lungs were characterized by alveolar inflammation (containing a mixed population of mononuclear cells, macrophages and/or granulocytes) associated with the presence of intra-alveolar granular material in all animals of the mid- and high-concentration main and recovery groups. Perivascular mononuclear inflammation was observed in (almost) all mid- and high-concentration main and recovery animals. Granulomata (micro granulomas) were found in the lungs of 14/20 animals of the high-concentration main group, and in 17/20 and 17/19 of the mid- and high-concentration recovery groups, respectively. The microscopic findings in the lungs were consistent with those described in the literature following inhalation of inert particles and can be attributed to macrophage overload with inhaled test material and subsequent incomplete clearance leading to macrophage accumulation in the terminal air spaces.

Microscopic accumulations of macrophages were observed in the tracheobronchial lymph nodes of the majority of animals of the mid- and high-concentration main groups, accompanied by lymphoid hyperplasia in some animals. An increased incidence of these lesions was seen in animals sacrificed at the end of the recovery period. The changes in the local lymph nodes were considered to be a secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

In addition to the above-described local toxicity, the extensive histopathological examination of organs and tissues did not reveal any indications for systemic toxicity in response to the exposure to the test material. In contrast to the evident pathology observed in the mid- and high-concentration groups, very limited changes were found in the low-concentration group. Histopathological examination of the lungs revealed a minimal amount of intra-alveolar matter in the lungs of only one animal of the low-concentration main group. Minimal alveolar inflammatory foci were observed in four animals of the low-concentration main group and ten animals of the recovery group. While not observed in concurrent controls, minimal to mild (multi- ) focal alveolitis can be observed in control animals as common background pathology. Given the absence of any further tissue reaction, these minimal findings were considered to be below a level of toxicological concern.

In conclusion, the results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 3.06 mg/m3. Systemic toxicity was not observed up to concentrations of 103.4 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

3.06 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

Subacute and suchronic inhalation toxicity studies available. The latest with 13 weeks of recovery is complete and sufficient to fulfill the REACh annex IX requirements

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

The substance was tested in three repeated-dose toxicity studies performed by the oral and inhalation routes:

Oral route

In a dose range-finding study performed in compliance with Good Laboratory Practice, the test substance was administered daily by gavage to CD rats for 14 days. Three groups, each comprising four male and four female rats received the test substance at doses of 100, 300 or 1,000 mg/kg bw/day.

During the study, the animals were checked for mortality, clinical condition, bodyweight, food consumption and water consumption. Animals were sacrificed on completion of the treatment period and a complete macroscopic post-mortem examination was performed. Selected organs were weighed and preserved.

There were no mortalities and the clinical condition of the animals was unaffected by treatment. Three females receiving 1000 mg/kg/day showed unremarkable bodyweight gains, but one female showed overall slight weight loss with an uncertain relationship to the treatment.  Overall bodyweight gain in males was similar in all groups. Food consumption in males was unaffected by treatment and there was no conclusive effect on the food intake of females. There were no effects of treatment on the organ weights of animals which received 100, 300 or 1000 mg/kg/day except a slightly lower liver weight in females that received 1,000 mg/kg bw/day. Macroscopic examination at necropsy after 14 days of treatment did not reveal any abnormalities.  

Based on these results, it could be concluded that the NOAEL for the test substance was 1000 mg/kg bw/day and dose levels of 100, 300 and 1000 mg/kg/day would be suitable for use in an associated main OECD 421 reproductive/developmental toxicity screening study.

Inhalation

In a pilot study according to OECD guideline 412 and performed in compliance with Good Laboratory Practice, the test substance was administered by nose-only inhalation exposure for 6 hours per day (5 days per week) to Wistar rats. Three groups, each comprising five male and five female rats received the test substance at achieved exposure levels of 100, 500 and 2020 mg/m3 on 10 occasions during a 14 day period. A similarly constituted control group received air at the same operating conditions as the other groups.

During the study, the animals were checked for mortality, clinical condition, bodyweight and food consumption. At necropsy, blood samples were collected and macroscopic examination was performed. Selected organs were weighed and an histopathological investigation was performed .

The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3 µm) for a repeat dose inhalation study. 

There was no mortality during the study. Clinical signs related to treatment were noted only in animals given 2020 mg/m3. All but one female and only 2 males had a transient slight increase in respiratory rate. In females, slight and in a few occasions moderate, laboured and/ or noisy respiration was observed throughout the study. In males, slight laboured respiration was noted only on Day 2 in 3 animals. Hunched posture was observed from Day 4 to Day 10 in 1 to 3 females and on Days 10-11 in 1 male. In addition, sneezing was noted for two females on Day 10 and 11. 

Changes in bodyweight and bodyweight gain were noted only in animals that received 2020 mg/m3 . Temporary mean bodyweight loss was observed at the beginning of the study in both sexes on (Days 1-4) and correlated with the significant decrease in food consumption noted during the same treatment-period. In addition, females showed a statistically significant decrease in mean bodyweight gain on the overall period of treatment (-36% compared with controls).

Following 2 weeks of treatment, an increase in the white blood cell count was observed in both sexes at all doses but without dose-dependency. In addition, a statistically significant increase of relative Neutrophils and concomitant decrease of relative Lymphocytes were observed in males given 2020 mg/m3 and in all treated females. These changes in haematology might be contributed to changes in the lungs.

Macroscopic examination revealed enlargement and/or pale discoloration of the lungs in animals given 500 or 2020 mg/m3. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels. There was a clear relationship between these microscopic findings and the dose. The alterations in the lungs were characterized by the presence of white/pink foreign material in the alveoli. The presence of foreign alveolar material was generally minimal at the low dose and moderate at the high dose. The inflammatory reactions in the lungs ranged from minimal to moderate severity, with the presence of a mixed cell population in the bronchiolar and alveolar compartments. The incidence and severity of the inflammatory responses were generally increased consistently with the dose. The formation of microgranulomas was observed microscopically in the alveoli of animals treated at a dose level of 2020 mg/m3. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in the Low dose, increased (mild) in the Mid dose and moderate in the High dose group.

The mean absolute and relative lung weights were significantly higher in males and females at dose levels of 100, 500 and 2020 mg/m3 compared to the controls.

Based on these findings, it was concluded that the LOAEL of 100 mg/m3 could be the maximum target exposure level that would be tolerated for a 90 day exposure in the same species.

The toxicity of the test substance was subsequently investigated in a 90-day sub-chronic study performed in Wistar rats according to OECD 413 and Good Laboratory Practices.

Four main groups of ten male and ten female rats each were exposed nose-only to target concentrations of 0 (control), 3, 20 or 100 mg/m3 for 6 hours/day, 5 days/week over a 90-day period. Animals of the main groups were sacrificed on the day after the last exposure. In addition, recovery groups – also consisting of ten male and ten female animals each – were simultaneously exposed with animals of the main groups and were sacrificed after a 13-week recovery period following the last exposure.

Endpoints to assess toxicity included clinical and ophthalmoscopic observations, growth, food consumption, haematology, clinical chemistry and organ weights. In addition, the animals were macroscopically examined at necropsy, the right lung lobes were lavaged and used for determination of biochemical markers and cell differentials, and the left lung lobes together with the full respiratory tract and a large number of organs and tissues were examined microscopically.

The average Mass Median Aerodynamic Diameter of the particles in the test atmosphere was 2.38, 2.42 and 2.15 µm for the low-, mid- and high-concentration groups, respectively while the achieved aerosol concentrations for the corresponding groups were 3.06, 20.5 and 103.4 mg/m3. Chemical analysis of test atmosphere samples by UPLC-MS/MS indicated that the composition of the material in the test atmospheres was similar to the original material and was stable across the exposure period.

Exposure-related mortality did not occur during the study. Clinical and ophthalmoscopic observations revealed no exposure-related abnormalities. Growth and food consumption were not adversely affected by the exposure and were comparable across the groups.

Analysis of haematology parameters revealed an increase in neutrophil numbers in blood of animals of the mid- and high- concentration group at the end of the exposure period, which was considered to be related to the inflammatory changes in the lower airways. Consequently, total white blood cells were increased in males of the high-concentration group, and the relative contribution of lymphocytes was decreased in females of the mid- and high concentration and in males of the high-concentration group. At the end of the recovery period, these changes in total and differential white blood cells were still found. Exposure to the test material did not result in any changes in clinical chemistry parameters.

Analysis of bronchoalveolar lavage (BAL) parameters revealed a concentration-related increase in total protein, phospholipids and lactate dehydrogenase activity, and increased numbers of (mainly) neutrophils and macrophages in BAL fluid of animals of the mid- and high concentration main groups. Lymphocyte numbers were also slightly increased in females of the mid- and high concentration and males of the high-concentration main groups. Consequently, the percentage distribution of white blood cells in BAL fluid shifted from ~100% macrophages – which is normally observed in healthy animals – towards an increased contribution of primarily neutrophils in animals of the mid- and high-concentration group. Comparable changes were generally still observed in animals sacrificed at the end of the recovery period.

Organ weight data showed an increase in absolute and relative weight of the lungs in animals of the mid- and high-concentration group at the end of the exposure period, and at the end of the recovery period.On average, relative lung weights were increased by 29% (males) to 41% (females) at the mid-concentration, and by 131% (females) to 135% (males) at the high-concentration level which was a indicative of a pulmonary overload.

Macroscopic examination at scheduled termination revealed white spots on the lungs of 12/20 animals of the high-concentration main group while at the end of the recovery period

all animals of the high-concentration recovery group and 10/20 animals of the mid-concentration recovery group showed white spots.

In addition, enlargement of the tracheobronchial lymph nodes was found in 6/20 and 12/20 animals of the mid- and high concentration main groups, and in 17/20 and 19/19 (note that one male was humanely sacrificed before scheduled necropsy) of the mid- and high-concentration recovery groups, respectively.

Microscopic examination revealed exposure-related histopathological changes in the nose, larynx, lungs and tracheobronchial lymph nodes of animals of the mid- and high-concentration main groups.

The changes in the nose were characterized by an increased presence of hyaline droplets in the olfactory epithelium in the caudal parts of the nasal tissues. The incidence of this finding, observed in several animals at the end of the exposure period, was slightly higher at the end of the recovery period.

Histopathological changes in the larynx consisted of focal squamous metaplasia of the lining epithelium, observed in 1/20 and 8/20 animals of the mid- and high-concentration main groups, respectively. No exposure-related laryngeal abnormalities were found at the end of the recovery period.

The changes in the lungs were characterized by alveolar inflammation (containing a mixed population of mononuclear cells, macrophages and/or granulocytes) associated with the presence of intra-alveolar granular material in all animals of the mid- and high-concentration main and recovery groups. Perivascular mononuclear inflammation was observed in (almost) all mid- and high-concentration main and recovery animals. Granulomata (micro granulomas) were found in the lungs of 14/20 animals of the high-concentration main group, and in 17/20 and 17/19 of the mid- and high-concentration recovery groups, respectively. The microscopic findings in the lungs were consistent with those described in the literature following inhalation of inert particles and can be attributed to macrophage overload with inhaled test material and subsequent incomplete clearance leading to macrophage accumulation in the terminal air spaces.

Microscopic accumulations of macrophages were observed in the tracheobronchial lymph nodes of the majority of animals of the mid- and high-concentration main groups, accompanied by lymphoid hyperplasia in some animals. An increased incidence of these lesions was seen in animals sacrificed at the end of the recovery period.The changes in the local lymph nodes were considered to be a secondary response to the increased influx of macrophages clearing the test material from the terminal airways and alveolar spaces and trafficking to the local draining lymph nodes.

In addition to the above-described local toxicity, the extensive histopathological examination of organs and tissues did not reveal any indications for systemic toxicity in response to the exposure to the test material. In contrast to the evident pathology observed in the mid- and high-concentration groups, very limited changes were found in the low-concentration group. Histopathological examination of the lungs revealed a minimal amount of intra-alveolar matter in the lungs of only one animal of the low-concentration main group. Minimal alveolar inflammatory foci were observed in four animals of the low-concentration main group and ten animals of the recovery group. While not observed in concurrent controls, minimal to mild (multi- ) focal alveolitis can be observed in control animals as common background pathology. Given the absence of any further tissue reaction, these minimal findings were considered to be below a level of toxicological concern.

In conclusion, the results from the bronchoalveolar lavage fluid and haematology correlated with the histopathological findings and were suggestive of lung injury consistent with the inhalation of in poorly soluble particulate matter. Since no toxicologically relevant adverse changes were observed in the low-concentration group, the NOAEC for sub-chronic inhalation exposure to was placed at 3.06 mg/m3. Systemic toxicity was not observed up to concentrations of 103.4 mg/m3.

Justification for classification or non-classification

No classification for repeated dose toxicity is warranted according to regulation (EC) No. 1272/2008 and its subsequent amendments on classification, labeling and packaging (CLP) of substances and mixtures.