Reaction mass of N,N’-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide), Octadecanamide, 12-hydroxy-N-[2-[(1-oxooctadecyl)amino]ethyl]- and Octadecanoic acid, 12-hydroxy-, 1-hexyl-12-[[2-[(12-hydroxy-1-oxooctadecyl)amino]ethyl]amino]-12-oxododecyl ester

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

From 06 November 2012 to 14 February 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across study hence maximum reliability rating of 2 assigned according to ECHA guidance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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 a the future 90-day inhalation study.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

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

IUPAC Name:

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

Constituent 2

Reference substance name:

100545-48-0

Cas Number:

100545-48-0

IUPAC Name:

100545-48-0

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material: Octadecanoic acid, 12-hydroxy-, reaction products with ethylenediamine

Test animals

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.

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

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 style (TSE Systems GmbH, Bad Homburg, Germany). Samples were collected at least once during each week of exposure for each concentration tested. Samples were 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, 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 these 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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0.1; 0.5 and 2.0 mg/L
Basis:
other: target concentrations

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.10 ; 0.50 and 2,02 mg/L
Basis:
analytical conc.

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

Examinations

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: Body weight of each animal was recorded with 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 abnormality 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 normal 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.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the animals exposed to the concentrations of 2.02 mg/L, the following clinical signs were observed:
Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 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.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 on the overall period of treatment (-36% compared with controls).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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.

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

Description (incidence and severity):

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. Slight decreased of Red Blood Count and 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 biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

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 pathological findings:

effects observed, treatment-related

Description (incidence and severity):

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 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 females of the 0.50 mg/L group.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Substance-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 as presence of white/pink foreign material in the alveoli, 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 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 appeared 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 moderateat 2.02 mg/L (see table 7.5.2/3).

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:
• Slight increase in respiratory rate was observed in 4/5 females on Day 1 and in 2/5 males on Days 3-4,
• Slight and in few occasion moderate laboured respiration was noted throughout the study in females. In males, a slight laboured respiration was observed on Day 2 in 3/5 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 on 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. Slight decreased of Red Blood Count and 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 5/5 males and 5/5 females in the 2.02 mg/L group. Pale foci were seen in 2/5 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 as presence of white/pink foreign material in the alveoli, 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 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 appeared 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 moderateat 2.02 mg/L (see table 7.5.2/3).

Effect levels

Key result

Dose descriptor:

LOAEC

Effect level:

0.1 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: At 0.1 mg/L, 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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Exposure to the test substance at gravimetrically determined dose levels of 0.10, 0.50 and 2.02 mg/L 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 mixed cell population. Also, foreign material was seen in the alveoli. The incidence and severity of the inflammatory responses and 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 Practices, 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 target exposure levels of of 0.1, 0.5 and 2.00 mg/L. A similarly constituted Control group received air at 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 achieved aerosol concentrations were 0.10, 0.50 and 2.02 mg/L (100, 100 and101% of target). 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 2.02 mg/L. 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, 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 2.02 mg/L . 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 2.02 mg/l and in all treated females. These changes in haematology might be contributed to changes in the lungs. Slight decreased of Red Blood Count and 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 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 0.50 or 2.02 mg/L. Treatment related histopathological changes were seen in lungs and tracheobronchial lymph nodes at all doses levels.There was clear relationship of these microscopic findings to the dose. The alterations in the lungs were characterized as 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 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 2.02 mg/L. A similar pattern of dose relationship was recorded for the accumulation of foamy macrophages in the tracheobronchial lymph nodes with low severity (minimal) in Low dose, increased (mild) in Mid dose and moderate in the High dose group. The mean absolute and relative lungs weights adjusted for both brain and terminal body weight were significantly higher in males and females at dose levels of 0.1, 0.5 and 2.02 mg/L compared to controls.

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