Alkanol reaction products with bis(aminoalkyl(C=1~6))carbomonocycle, alkoxy(C=3~8)alkanol(C=2~7) and 2,4-TDI

Endpoint summary

Description of key information

In a Dose Range-finding toxicity study performed by oral route, rats treated for 14 days showed no remarkable effects up to a dose-level of 1000 mg/kg bw/day.  

In a subacute inhalation toxicity study performed in accordance with OECD guideline No 412 and in compliance with GLP, the test substance was responsible for a mild pulmonary inflammation. The changes in lungs weight and macroscopic examination correlated with the microscopic findings. Based on these findings, the NOAEC was set to 31.3 mg/m3.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

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

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 september 2015 - 1 september 2016

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)

Deviations:

no

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: Envigo RMS (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 215 to 247 g (males) and 163 to 186 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum,Harlan Teklad 2014C Diet
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with 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:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.1 - <= 2.4 µm

Geometric standard deviation (GSD):

2.5

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 2.1 - 2.4 µm with Geometric Standard Deviation (GSD) of 2.50 - 3.08.
The mean Mass Median Aerodynamic Diameter values were within the ideal range of 1 to 3 µm indicating that the test substance was respirable to rat.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 19L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (20.8; 20.8; 21.0 and 21.2 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow was 19 L/minute.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). 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 < 0.42, 0.76, 1.27, 2.86, 4.9 and 8.0 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate of 20, 70, 50 and 20 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group.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.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with UV detection analytical method was used.

- 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 during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 5.86; 31.3 and 157 µg/L and corresponded to 98; 104 and 105% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days / week
6 hours daily exposure

Dose / conc.:

0.006 mg/L air (nominal)

Remarks:

Target exposure level for low-concentration group

Dose / conc.:

0.03 mg/L air (nominal)

Remarks:

Target exposure level for mid-concentration group

Dose / conc.:

0.15 mg/L air (nominal)

Remarks:

Target exposure level for high-concentration group

Dose / conc.:

0.006 mg/L air (analytical)

Remarks:

Achieved concentration for low-concentration group

Dose / conc.:

0.031 mg/L air (analytical)

Remarks:

Achieved concentration for mid-concentration group

Dose / conc.:

0.157 mg/L air (analytical)

Remarks:

Achieved concentration for high-concentration group

No. of animals per sex per dose:

3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 6, 30 or 150 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeated dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0.0393, 0.203 or 0.984 mg/L (39.3, 203 or 984 µg/L ) of the test substance. Histopathological treatment related findings were evident in the lungs and tracheobronchial and mediastinal lymph nodes. The incidence and severity of findings in the lungs of animals exposed to 0.984 mg/L were considered adverse and therefore this level was not suitable for a longer term study. Changes in the lung at 0.203 mg/L were generally of lower incidence and/or severity than those seen at 0.984 mg/L; but effects in females were genrally of higher incidence and/or severity than those in males, therefore for this study, a high exposure level targeted at 0.150 mg/L was anticipated to induce treatment related changes similar to those previously seen but expected to be tolerated for 4 weeks. Target exposure levels of 0.03 mg/L and 0.006 mg/L were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship..

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, detailed observations were recorded at the following times in relation to dose administration:
* Pre-exposure observation
* As each animal is returned to its home cage
* As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded twice during the week before treatment commenced, on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started, and each week throughout the treatment. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (RETA), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (0.984 mg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- all mediastinal lymph nodes showing macroscopic abnormality were examined,
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (0.00586 mg/L) and 3 (0.0313 mg/L).

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (0.157 mg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

Statistics:

All analyses were carried out using the individual animal as the basic experimental unit.
# The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For pathology if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a non statistically significant decrease in mean body weight gains at termination for both sexes exposed to 157 µg/L when compared with control (73% or 82% of control for males and females respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower group mean lymphocyte counts were evident in males and females exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control).
Mean white blood cells counts in both sexes exposed to 157 µg/L, were lower than control reaching statistical significance in females (0.79X or 0.72X control for males and females respectively) due to the lower lymphocyte counts but basophil and large unstained cell counts were also lower.

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

Description (incidence and severity):

Higher group mean lung and bronchi weights, adjusted for terminal body weight, were observed for both sexes exposed to 157 µg/L, 1.62X or 1.66X control for males and females respectively; and males exposed to 31.3 µg/L, 1.10X control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 157 µg/L, tracheobronchial lymph nodes were enlarged in three males and all females and pale colour was also observed in one male and four females.
At 157 µg/L, mediastinal lymph nodes were enlarged in two males and all females and pale colour was seen in one male and all females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment were present in the lungs of all animals that received 157 µg/L. Treatment related changes were also seen in all the males and the majority of females that received 31.3 µg/L. Changes observed at 31.3 µg/L were considered to be a normal physiological response and not adverse.
An increase in cellularity of the paracortex was present in the tracheobronchial lymph nodes of 2 males and 4 females that were treated with 157 µg/L.
An increase in cellularity of the paracortex was present in the mediastinal lymph nodes of 1 male and all females that received 157 µg/L

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The cellularity, distribution and morphology of the bone marrow were unaffected by the treatment.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities and no test article-related clinical signs during the study.

BODY WEIGHT AND WEIGHT GAIN (cf table 7.5.2/1 in chapter any other information on results)
Mean body weight gains at termination were lower for both sexes exposed to 157 µg/L when compared with control (73% or 82% of control for males and females respectively).
Initial group mean body weight losses were apparent for male treated groups at the mid-week occasion during Week 1, however, a similar effect was not evident for female treated groups, losses were small and Week 2 group means were similar for all male groups, including control. The weight loss apparent at the mid-week occasion of Week 4 (Day 25) for all groups was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry.
There were no test article-related effects on bodyweight or body weight gain at 5.78 or 31.3 µg/L after 4 weeks of treatment.

FOOD CONSUMPTION
There were no treatment related changes on food consumption.

HAEMATOLOGY
Lower group mean lymphocyte counts were evident in males and females exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control). Mean white blood cells counts in both sexes exposed to 157 µg/L, were lower than control reaching statistical significance in females (0.79X or 0.72X control for males and females respectively) due to the lower lymphocyte counts but basophil and large unstained cell counts were also lower.
This decrease in mean white blood cell counts was likely to be related to margination of these cells in the lung as part of the inflammatory response; however, it may also be related to non-specific stress or chemical toxicity as rodents are especially susceptible to stress-induced leukopenia

CLINICAL CHEMISTRY
There were no test article-related effects.

ORGAN WEIGHTS (cf table 7.5.2/2 in chapter any other information on results)
Higher group mean lung and bronchi weights, adjusted for terminal body weight, were observed for both sexes exposed to 157 µg/L, 1.62X or 1.66X control for males and females respectively; and males exposed to 31.3 µg/L, 1.10X control.

GROSS PATHOLOGY (cf table 7.5.2/3 in chapter any other information on results)
At 157 µg/L, tracheobronchial lymph nodes were enlarged in three males and all females and pale colour was also observed in one male and four females.
At 31.3 µg/L, one male and one female showed pale and enlarged tracheobronchial lymph nodes but no microscopic changes correlated with these findings.
At 157 µg/L, mediastinal lymph nodes were enlarged in two males and all females and pale colour was seen in one male and all females.
At 31.3 µg/L, the female with pale and enlarged tracheobronchial lymph nodes also showed pale colour and enlargement of the mediastinal lymph node but nomicroscopic changes correlated with these findings.


HISTOPATHOLOGY: NON-NEOPLASTIC (cf table 7.5.2/4 in chapter any other information on results)
Histopathological findings related to treatment were seen in the lungs, tracheobronchial and mediastinal lymph nodes:

in the lungs, changes related to treatment were present in all animals that received 157 µg/L. Increased numbers of macrophages were present within the alveoli and alveolar ducts. Macrophages were often hypertrophic with foamy cytoplasm and were accompanied by an infiltrate of neutrophils and type II pneumocyte hyperplasia at the bronchoalveolar junction, consistent with an inflammatory response in the alveoli and alveolar ducts. Eosinophilic material was present diffusely in the alveoli and alveolar ducts and had a granular appearance. An infiltrate of inflammatory cells consisting of mononuclear cells was present in predominantly the perivascular region but was also peribronchiolar in some cases. These changes were consistent with a reactive inflammatory response secondary to inhalation of the test item and attempted clearance of the inhaled material by the immune system. A relationship to dose was present, with minimal changes present in the lungs of animals that received 31.3 µg/L. The findings in the lungs were confined to a minimal macrophage response within the alveoli and alveolar ducts at 31.3 µg/L. These findings were considered to be a normal physiological response and not adverse. No changes were apparent in the lungs of animals that received 5.86 µg/L. The observed microscopic changes and the likely accumulation of test item in the lungs correlate with the higher group mean lung and bronchi weights observed in animals that received 31.3 or 157 µg/L.
Enlargement and abnormal colour of the tracheobronchial and mediastinal lymph nodes was present predominantly in animals that received 157 µg/L. The lymph node enlargement correlated microscopically with an increase in cellularity of the paracortex. These organs receive lymphatic drainage from tissues within the thoracic cavity, including the lungs and bronchi, and therefore enlargement and increase in paracortical cellularity are likely to reflect a response to the inflammatory process present in the lungs and bronchi.

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow were unaffected by the treatment.

Key result

Dose descriptor:

NOEC

Effect level:

0.031 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

no

7.5.2 /1 Body weight and body weight change

Group / sex		Day									Change 1-29
		1	4	8	11	15	18	22	25	29
1F	Mean	172	174	179	180	186	186	189	183	193	20
	SD	7.0	6.7	7.0	5.1	5.7	5.2	6.5	7.3	11.2	9.3
	N	5	5	5	5	5	5	5	5	5	5
2F	Mean	174	176	182	182	191	191	194	187	196	22
	SD	5.8	6.9	7.9	5.4	6.4	7.8	6.8	8.7	8.2	5.0
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										108
3F	Mean	176	176	185	184	191	192	198	191	204	27
	SD	8.6	7.5	9.7	11.8	11.2	8.1	8.1	9.4	9.2	3.8
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										134
4F	Mean	177	177	182	180	185	189	193	182	194	17
	SD	7.0	7.5	9.3	10.6	7.7	11.8	11.4	10.3	10.8	7.6
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										82 NS

 

Group / sex		Day									Change 1-29
		1	4	8	11	15	18	22	25	29
1M	Mean	232	232	243	248	260	266	274	267	285	53
	SD	8.8	8.2	10.8	12.4	15.3	14.8	16.2	13.4	17.6	12.4
	N	5	5	5	5	5	5	5	5	5	5
2M	Mean	230	232	243	244	258	259	269	263	279	49
	SD	2.8	7.0	6.9	9.1	7.6	9.7	8.8	11.8	9.8	8.3
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										93
3M	Mean	237	233	245	251	264	265	276	266	286	49
	SD	5.8	3.4	5.7	5.3	6.8	7..7	10.5	11.4	10.5	11.1
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										93
4M	Mean	228	226	238	237	252	251	259	245	267	39
	SD	10.2	10.9	12.0	16.7	16.1	18.1	20.3	21.1	24.2	15.8
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										73 NS

 

	Control	Test substance
Dose group	1	2	3	4
Dose (µg/l)	0	5.86	31.3	157

 

M= Male F= female SD= Standard Deviation N= number of animals examined

** p < 0.01 * p< 0.0.5  NS: Non Significant

 

7.5.2 /2 Lungs and Bronchi weights- group mean absolute and adjusted values (g) for animals killed after 4 weeks of treatment

Group / sex		Terminal Body weight (g)	Lungs and Bronchi weight (g)	Adjusted Lungs and Bronchi weight (g)
1F	Mean	194	0.981	1.003
	SD	11	0.077
	N	5	5
2F	Mean	196	0.934	0.940
	SD	9	0.058
	N	5	5
3F	Mean	204	1.054	1.006
	SD	10	0.071
	N	5	5
4F	Mean	194	1.651	1.671 **
	SD	11	0.230
	N	5	5

 

Group / sex		Terminal Body weight (g)	Lungs and Bronchi weight (g)	Adjusted Lungs and Bronchi weight (g)
1M	Mean	286	1.151	1.126
	SD	18	0.128
	N	5	5
2M	Mean	280	1.162	1.164
	SD	10	0.081
	N	5	5
3M	Mean	287	1.270	1.239*
	SD	11	0.058
	N	5	5
4M	Mean	267	1.772	1.825 **
	SD	23	0.098
	N	5	5

 

	Control	Test substance
Dose group	1	2	3	4
Dose (µg/l)	0	5.86	31.3	157

 

M= Male F= female SD= Standard Deviation N= number of animals examined

** p < 0.01 * p< 0.0.5

 

7.5.2 /3 Macropathology results

Summary of findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Enlarged	0	0	1*	3	0	0	1*	5
Abnormal colour (pale)	0	0	1*	1	0	0	1*	4

Summary of findings in the mediastinal lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Enlarged	0	0	0	2	0	0	1*	5
Abnormal colour (pale)	0	0	0	1	0	0	1*	5

* No microscopic changes correlated with these findings

7.5.2 /4 Histopathology results

Lungs

 

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Alveolar Macrophages Increased, Minimal Slight Moderate	0 0 0	0 0 0	5 0 0	0 1 4	0 0 0	0 0 0	4 0 0	0 2 3
Total	0	0	5	5	0	0	4	5
Inflammation, Alveoli Slight	0	0	0	5	0	0	0	5
Total	0	0	0	5	0	0	0	5
Infiltrate, inflammatory cell, perivascular Minimal Slight	0 0	0 0	0 0	4 1	0 0	0 0	0 0	5 0
Total	0	0	0	5	0	0	0	5
Eosinophilic material- alveolar Slight Moderate	0 0	0 0	0 0	1 4	0 0	0 0	0 0	1 4
Total	0	0	0	5	0	0	0	5

 

Tracheobronchial lymph nodes

 

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Increased Cellularity, paracortex Minimal Slight	0 0	0 0	0 0	2 0	0 0	0 0	0 0	3 1
Total	0	0	0	2	0	0	0	4

 

Mediastinal lymph nodes

 

Summary of treatment related findings in the mediastinal lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	0	0	0	2	0	0	1	5
Increased Cellularity, paracortex Minimal	0	0	0	1	0	0	0	5
Total	0	0	0	1	0	0	0	5

 

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 5.86, 31.3 or 157 µg/L. Changes related to treatment with the test substance were seen in the lungs, tracheobronchial and mediastinal lymph nodes.
In the lungs these changes consisted of eosinophilic granular material within the alveoli and alveolar ducts of animals that received 157 µg/L; associated with this was the presence of increased numbers of macrophages and a neutrophilic infiltrate. Hyperplasia of the type II pneumocytes at the junction of the alveolar ducts and alveoli and perivascular mononuclear cell infiltrate was also evident. The combination of findings occurring in the lungs of animals that received 157 µg/L, together with lower body weight gain in that group, was such that the lung findings in this dose group were considered adverse.
Increased cellularity of the paracortex of the tracheobronchial and mediastinal lymph nodes were apparent, predominantly in animals that received 157 µg/L and 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.
On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 31.3 µg/L.

Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks. The study was designated to provide a rational basis for the assessment of the toxicological risk to man.

Three groups, each comprising five male and five female rats, received the test item at target exposure levels of 6, 30 or 150 µg/L. A similarly constituted control group received air at the same operating conditions as the 150 µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 5.86, 31.3 and 157 µg/L (98, 104 and 105% of the target concentrations). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

There were no test article-related deaths or effects on clinical signs, food consumption, haematology (bone marrow) or blood chemistry. Mean body weight gains at termination were lower for males and females exposed to 157 µg/L when compared with control (73% and 82% of control for males and females respectively). A similar effect was not apparent at the lower exposure levels.

Haematology showed lower group mean lymphocyte counts in both sexes exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control). Lower mean white blood cells counts were evident in both sexes exposed to 157 µg/L, reaching statistical significance in females (0.79X or 0.72X control for males and females respectively). Lower basophil and large unstained cell counts were also apparent; there were no effects in animals exposed to 31.3 or 5.86 µg/L.

Higher group mean lung and bronchi weights were observed for males and females exposed to 157 µg/L and males exposed to 31.3 µg/L; up to 1.62X control for males and 1.66X control for females (values adjusted for terminal body weight). There were no effects in animals exposed to 5.86 µg/L.

Macroscopic examination revealed enlargement of the tracheobronchial lymph nodes in the majority of animals exposed to 157 µg/L and one animal of each sex exposed to 31.3 µg/L. Enlarged mediastinal lymph nodes were apparent for two males and all females exposed to 157 µg/L and one female exposed to 31.3 µg/L. Lymph node enlargement was often accompanied by pallor. No microscopic findings associated with the enlarged lymph nodeswere seen in the two animals at 31.3 µg/L. There were no macroscopic effects in animals exposed to 5.86 µg/L.

Microscopically, changes related to treatment were seen in the lungs, tracheobronchial and mediastinal lymph nodes. In the lungs, changes related to treatment were present in the lungs of all animals that received 157 µg/L. Increased numbers of macrophages were present within the alveoli and alveolar ducts and were often hypertrophic with foamy cytoplasm and were accompanied by an infiltrate of neutrophils and type II pneumocyte hyperplasia at the bronchoalveolar junction. Eosinophilic material was present diffusely in the alveoli and alveolar ducts and had a granular appearance. An infiltrate of inflammatory cells consisting of mononuclear cells was present in predominantly the perivascular region but was also peribronchiolar in some cases. Treatment related changes were also seen in all the males and the majority of females that received 31.3 µg/L. The findings in the lungs were confined to a minimal macrophage response within the alveoli and alveolar ducts at 31.3 µg/L. These findings were considered to be a normal physiological response following inhalation of particle matter and were not adverse. The increase in macrophages was associated with the presence of eosinophilic granular material in the lungs (likely the test item) and was considered to be related to pulmonary clearance. There were no effects in animals exposed in 5.86 µg/L. In the tracheobronchial lymph nodes an increase in cellularity of the paracortex was evident in two males and four females exposed to 157 µg/L. An increase in cellularity of the paracortex was present in the mediastinal lymph nodes of one male and all females exposed to 157 µg/L.These findings 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 31.3 µg/L.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

157 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII requirements

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 september 2015 - 1 september 2016

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)

Deviations:

no

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: Envigo RMS (UK) Ltd.
- Age at study initiation:61 to 67 days
- Weight at study initiation: 215 to 247 g (males) and 163 to 186 g (females)
- Fasting period before study:no
- Housing: The animals were housed five of one sex per cage
- Diet: Ad libitum,Harlan Teklad 2014C Diet
- Water: Ad libitum, potable water taken from the public supply.
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 23°C
- Humidity: 40 to 70%
- Air changes (per hr):Each animal room was supplied with 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:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.1 - <= 2.4 µm

Geometric standard deviation (GSD):

2.5

Remarks on MMAD:

MMAD / GSD: The Mass Median Aerodynamic Diameter (MMAD) of the test atmosphere of all groups was in the range of 2.1 - 2.4 µm with Geometric Standard Deviation (GSD) of 2.50 - 3.08.
The mean Mass Median Aerodynamic Diameter values were within the ideal range of 1 to 3 µm indicating that the test substance was respirable to rat.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow through nose only chamber. This system was an aluminium alloy construction comprising a base unit, three animal exposure sections and a top section.
- Method of holding animals in test chamber: Rats were held in polycarbonate tubes with their snouts protruding from the end of the tubes into the exposure chamber.
- Source and rate of air: From in-house compressed air system(breathing quality), Generator flow: 19L/minute
- System of generating particulates/aerosols: Wright Dust Feed mechanism designed to produce and maintain atmospheres of dust by suspending material scraped from the surface of a compressed powder in a stream of dry air. The concentrations of dust were altered by changing the gear ratio (and therefore the speed of rotation of the compressed powder towards the scraper blade) of the mechanism.
- Temperature, humidity, pressure in air chamber:
Temperature was measured using an electronic thermometer inserted into the inhalation chamber. The mean chamber temperatures were all within expected ranges (20.8; 20.8; 21.0 and 21.2 °C for controls, low, mid and high-dosed groups).
Humidity and pressure in air chamber were not reported.
- Air flow rate: Airflow was 19 L/minute.
- Method of particle size determination: Particle size analysis of generated atmospheres was performed using a 6-stage cascade impactor (Marple 296). 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 < 0.42, 0.76, 1.27, 2.86, 4.9 and 8.0 µm (aerodynamic diameter) was calculated. From this data, the Mass Median Aerodynamic Diameter (MMAD), and Geometric Standard Deviation (GSD) were calculated.
- Treatment of exhaust air: Extract airflow was drawn by in-house vacuum system at a flow rate of 20, 70, 50 and 20 L/minute for the control, low, mid and high-dose groups respectively. the airflow was filtered locally.

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 a glass fibre filters. Sampling was performed at least three times during each exposure and for each dose-group.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.
In addition, the aerosol concentrations measured by gravimetric analysis were checked by a chemical analysis once every week. High Performance Liquid Chromatography with UV detection analytical method was used.

- 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 during each exposure by gravimetrical analysis of the test item deposited on a sampling filter. Once every week, the gravimetric analysis was coupled to analytical analysis by HPLC with UV detection to check the accuracy of the gravimetric method.
The mean achieved concentrations were 5.86; 31.3 and 157 µg/L and corresponded to 98; 104 and 105% of the target concentrations respectively. The mean exposure concentrations measured by chemical and gravimetric analysis correlated well.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

5 days / week
6 hours daily exposure

Dose / conc.:

0.006 mg/L air (nominal)

Remarks:

Target exposure level for low-concentration group

Dose / conc.:

0.03 mg/L air (nominal)

Remarks:

Target exposure level for mid-concentration group

Dose / conc.:

0.15 mg/L air (nominal)

Remarks:

Target exposure level for high-concentration group

Dose / conc.:

0.006 mg/L air (analytical)

Remarks:

Achieved concentration for low-concentration group

Dose / conc.:

0.031 mg/L air (analytical)

Remarks:

Achieved concentration for mid-concentration group

Dose / conc.:

0.157 mg/L air (analytical)

Remarks:

Achieved concentration for high-concentration group

No. of animals per sex per dose:

3 groups, each comprising 5 male and 5 female rats received the test substance at target exposure levels of 6, 30 or 150 µg/L. A similarly constituted Control group received air only, at the same operating conditions as the high dose group.

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale:
In a previously conducted two-week repeated dose inhalation toxicity study, rats were exposed 5 days/week, 6 hours/day to 0.0393, 0.203 or 0.984 mg/L (39.3, 203 or 984 µg/L ) of the test substance. Histopathological treatment related findings were evident in the lungs and tracheobronchial and mediastinal lymph nodes. The incidence and severity of findings in the lungs of animals exposed to 0.984 mg/L were considered adverse and therefore this level was not suitable for a longer term study. Changes in the lung at 0.203 mg/L were generally of lower incidence and/or severity than those seen at 0.984 mg/L; but effects in females were genrally of higher incidence and/or severity than those in males, therefore for this study, a high exposure level targeted at 0.150 mg/L was anticipated to induce treatment related changes similar to those previously seen but expected to be tolerated for 4 weeks. Target exposure levels of 0.03 mg/L and 0.006 mg/L were selected for the intermediate and low groups respectively to identify a no-observed adverse effect level and to explore any possible dose relationship..

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Cages were inspected daily for evidence of ill-health amongst the occupants.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 4 of treatment, detailed observations were recorded at the following times in relation to dose administration:
* Pre-exposure observation
* As each animal is returned to its home cage
* As late as possible in the working day

BODY WEIGHT: Yes
- Time schedule for examinations:The weight of each animal was recorded twice during the week before treatment commenced, on the day that treatment commenced (Day 1), twice weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes
-The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started, and each week throughout the treatment. From these records the mean weekly consumption per animal (g/animal/week) was calculated for each cage.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40.
- Parameters examined: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Reticulocyte count (RETA), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count including Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PTP) and Activated partial thromboplastin time (APTT).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: During Week 4 of treatment and before dosing for all study animals.
- Animals fasted: Yes, overnight withdrawal of food.
- How many animals: 40
- Parameters examined: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Total bilirubin (Bili), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All study animals were sacrificed following 4 weeks of exposures.
GROSS PATHOLOGY: Yes
All animals were subject to a detailed necropsy. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded.
HISTOPATHOLOGY: Yes
The following tissues were examined for all study animals of Groups 1 (Control) and 4 (0.984 mg/L) sacrificed on completion of the 4-week treatment period:
Adrenals - cortex and medulla
Brain - cerebellum, cerebrum and midbrain
Heart - included auricular and ventricular regions
Kidneys - included cortex, medulla and papilla regions
Larynx - 5 sections
Liver - section from two main lobes
Lungs - section from all major lobes, to include bronchi
Nasal turbinates -4 levels including nasal cavity, paranasal sinuses, nasopharynx and nasopharynx duct and nasal associated lymphoid tissue
Oesophagus
Ovaries
Seminal vesicles
Spinal cord - transverse and longitudinal section at the cervical, lumbar and thoracic levels
Spleen
Stomach - included keratinised, glandular and antrum in sections
Testes
Thymus
Thyroid - included parathyroids in section where possible
Trachea - including bifurcation
Uterus - uterine body with cervix section
In addition:
- all mediastinal lymph nodes showing macroscopic abnormality were examined,
- Lungs, larynx, trachea (including bifurcation), nasal turbinates and tracheobronchial lymph nodes were examined for all study animals of groups 2 (0.00586 mg/L) and 3 (0.0313 mg/L).

Other examinations:

- HAEMATOLOGY, BONE MARROW:
Bone marrow smears were prepared immediately following death on completion of the scheduled treatment period. The smears from all animals of Groups 1 (Control) and 4 (0.157 mg/L) were examined to assess the cellularity, distribution and morphology of the marrow. The smears from animals of the intermediate and low dose groups were not examined.

- ORGAN WEIGHTS:
The following organs, taken from each animal killed after 4 weeks of treatment were weighted: Adrenals, Brain, Heart, Kidneys, Liver, Lungs with mainstem bronchi, Spleen, Testes and Thymus.
Bilateral organs were weighed together. Organ weights were also adjusted for terminal bodyweight, using the weight recorded before necropsy.

Statistics:

All analyses were carried out using the individual animal as the basic experimental unit.
# The following sequence of statistical tests was used for bodyweight, organ weight and clinical pathology:
A parametric analysis was performed if Bartlett's test for variance homogeneity was not significant at the 1% level. The F1 approximate test was applied. If the F1 approximate test was not significant at the 1% level, Williams' test was applied. If the F1 approximate test was significant, suggesting that the dose response was not monotone, Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test was still significant at the 1% level following both logarithmic and square-root transformations. The H1 approximate test was applied. If the H1 approximate test was not significant at the 1% level, Shirley's test was applied. If the H1 approximate test was significant, suggesting that the dose-response was not monotone, Steel's test was performed instead.
For pathology if 75% of the data (across all groups) were the same value, for example c, Fisher’s Exact tests were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.
# For organ weight data, analysis of covariance was performed using terminal bodyweight as covariate, unless non-parametric methods were applied.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was a non statistically significant decrease in mean body weight gains at termination for both sexes exposed to 157 µg/L when compared with control (73% or 82% of control for males and females respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower group mean lymphocyte counts were evident in males and females exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control).
Mean white blood cells counts in both sexes exposed to 157 µg/L, were lower than control reaching statistical significance in females (0.79X or 0.72X control for males and females respectively) due to the lower lymphocyte counts but basophil and large unstained cell counts were also lower.

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

Description (incidence and severity):

Higher group mean lung and bronchi weights, adjusted for terminal body weight, were observed for both sexes exposed to 157 µg/L, 1.62X or 1.66X control for males and females respectively; and males exposed to 31.3 µg/L, 1.10X control.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 157 µg/L, tracheobronchial lymph nodes were enlarged in three males and all females and pale colour was also observed in one male and four females.
At 157 µg/L, mediastinal lymph nodes were enlarged in two males and all females and pale colour was seen in one male and all females.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment were present in the lungs of all animals that received 157 µg/L. Treatment related changes were also seen in all the males and the majority of females that received 31.3 µg/L. Changes observed at 31.3 µg/L were considered to be a normal physiological response and not adverse.
An increase in cellularity of the paracortex was present in the tracheobronchial lymph nodes of 2 males and 4 females that were treated with 157 µg/L.
An increase in cellularity of the paracortex was present in the mediastinal lymph nodes of 1 male and all females that received 157 µg/L

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

The cellularity, distribution and morphology of the bone marrow were unaffected by the treatment.

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no mortalities and no test article-related clinical signs during the study.

BODY WEIGHT AND WEIGHT GAIN (cf table 7.5.2/1 in chapter any other information on results)
Mean body weight gains at termination were lower for both sexes exposed to 157 µg/L when compared with control (73% or 82% of control for males and females respectively).
Initial group mean body weight losses were apparent for male treated groups at the mid-week occasion during Week 1, however, a similar effect was not evident for female treated groups, losses were small and Week 2 group means were similar for all male groups, including control. The weight loss apparent at the mid-week occasion of Week 4 (Day 25) for all groups was due to overnight food withdrawal before the blood sampling procedures for haematology and blood chemistry.
There were no test article-related effects on bodyweight or body weight gain at 5.78 or 31.3 µg/L after 4 weeks of treatment.

FOOD CONSUMPTION
There were no treatment related changes on food consumption.

HAEMATOLOGY
Lower group mean lymphocyte counts were evident in males and females exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control). Mean white blood cells counts in both sexes exposed to 157 µg/L, were lower than control reaching statistical significance in females (0.79X or 0.72X control for males and females respectively) due to the lower lymphocyte counts but basophil and large unstained cell counts were also lower.
This decrease in mean white blood cell counts was likely to be related to margination of these cells in the lung as part of the inflammatory response; however, it may also be related to non-specific stress or chemical toxicity as rodents are especially susceptible to stress-induced leukopenia

CLINICAL CHEMISTRY
There were no test article-related effects.

ORGAN WEIGHTS (cf table 7.5.2/2 in chapter any other information on results)
Higher group mean lung and bronchi weights, adjusted for terminal body weight, were observed for both sexes exposed to 157 µg/L, 1.62X or 1.66X control for males and females respectively; and males exposed to 31.3 µg/L, 1.10X control.

GROSS PATHOLOGY (cf table 7.5.2/3 in chapter any other information on results)
At 157 µg/L, tracheobronchial lymph nodes were enlarged in three males and all females and pale colour was also observed in one male and four females.
At 31.3 µg/L, one male and one female showed pale and enlarged tracheobronchial lymph nodes but no microscopic changes correlated with these findings.
At 157 µg/L, mediastinal lymph nodes were enlarged in two males and all females and pale colour was seen in one male and all females.
At 31.3 µg/L, the female with pale and enlarged tracheobronchial lymph nodes also showed pale colour and enlargement of the mediastinal lymph node but nomicroscopic changes correlated with these findings.


HISTOPATHOLOGY: NON-NEOPLASTIC (cf table 7.5.2/4 in chapter any other information on results)
Histopathological findings related to treatment were seen in the lungs, tracheobronchial and mediastinal lymph nodes:

in the lungs, changes related to treatment were present in all animals that received 157 µg/L. Increased numbers of macrophages were present within the alveoli and alveolar ducts. Macrophages were often hypertrophic with foamy cytoplasm and were accompanied by an infiltrate of neutrophils and type II pneumocyte hyperplasia at the bronchoalveolar junction, consistent with an inflammatory response in the alveoli and alveolar ducts. Eosinophilic material was present diffusely in the alveoli and alveolar ducts and had a granular appearance. An infiltrate of inflammatory cells consisting of mononuclear cells was present in predominantly the perivascular region but was also peribronchiolar in some cases. These changes were consistent with a reactive inflammatory response secondary to inhalation of the test item and attempted clearance of the inhaled material by the immune system. A relationship to dose was present, with minimal changes present in the lungs of animals that received 31.3 µg/L. The findings in the lungs were confined to a minimal macrophage response within the alveoli and alveolar ducts at 31.3 µg/L. These findings were considered to be a normal physiological response and not adverse. No changes were apparent in the lungs of animals that received 5.86 µg/L. The observed microscopic changes and the likely accumulation of test item in the lungs correlate with the higher group mean lung and bronchi weights observed in animals that received 31.3 or 157 µg/L.
Enlargement and abnormal colour of the tracheobronchial and mediastinal lymph nodes was present predominantly in animals that received 157 µg/L. The lymph node enlargement correlated microscopically with an increase in cellularity of the paracortex. These organs receive lymphatic drainage from tissues within the thoracic cavity, including the lungs and bronchi, and therefore enlargement and increase in paracortical cellularity are likely to reflect a response to the inflammatory process present in the lungs and bronchi.

OTHER FINDINGS
Bone marrow analysis:
The cellularity, distribution and morphology of the marrow were unaffected by the treatment.

Key result

Dose descriptor:

NOEC

Effect level:

0.031 mg/L air (analytical)

Based on:

test mat.

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

no

7.5.2 /1 Body weight and body weight change

Group / sex		Day									Change 1-29
		1	4	8	11	15	18	22	25	29
1F	Mean	172	174	179	180	186	186	189	183	193	20
	SD	7.0	6.7	7.0	5.1	5.7	5.2	6.5	7.3	11.2	9.3
	N	5	5	5	5	5	5	5	5	5	5
2F	Mean	174	176	182	182	191	191	194	187	196	22
	SD	5.8	6.9	7.9	5.4	6.4	7.8	6.8	8.7	8.2	5.0
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										108
3F	Mean	176	176	185	184	191	192	198	191	204	27
	SD	8.6	7.5	9.7	11.8	11.2	8.1	8.1	9.4	9.2	3.8
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										134
4F	Mean	177	177	182	180	185	189	193	182	194	17
	SD	7.0	7.5	9.3	10.6	7.7	11.8	11.4	10.3	10.8	7.6
	N	5	5	5	5	5	5	5	5	5	5
	% of 1F										82 NS

 

Group / sex		Day									Change 1-29
		1	4	8	11	15	18	22	25	29
1M	Mean	232	232	243	248	260	266	274	267	285	53
	SD	8.8	8.2	10.8	12.4	15.3	14.8	16.2	13.4	17.6	12.4
	N	5	5	5	5	5	5	5	5	5	5
2M	Mean	230	232	243	244	258	259	269	263	279	49
	SD	2.8	7.0	6.9	9.1	7.6	9.7	8.8	11.8	9.8	8.3
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										93
3M	Mean	237	233	245	251	264	265	276	266	286	49
	SD	5.8	3.4	5.7	5.3	6.8	7..7	10.5	11.4	10.5	11.1
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										93
4M	Mean	228	226	238	237	252	251	259	245	267	39
	SD	10.2	10.9	12.0	16.7	16.1	18.1	20.3	21.1	24.2	15.8
	N	5	5	5	5	5	5	5	5	5	5
	% of 1M										73 NS

 

	Control	Test substance
Dose group	1	2	3	4
Dose (µg/l)	0	5.86	31.3	157

 

M= Male F= female SD= Standard Deviation N= number of animals examined

** p < 0.01 * p< 0.0.5  NS: Non Significant

 

7.5.2 /2 Lungs and Bronchi weights- group mean absolute and adjusted values (g) for animals killed after 4 weeks of treatment

Group / sex		Terminal Body weight (g)	Lungs and Bronchi weight (g)	Adjusted Lungs and Bronchi weight (g)
1F	Mean	194	0.981	1.003
	SD	11	0.077
	N	5	5
2F	Mean	196	0.934	0.940
	SD	9	0.058
	N	5	5
3F	Mean	204	1.054	1.006
	SD	10	0.071
	N	5	5
4F	Mean	194	1.651	1.671 **
	SD	11	0.230
	N	5	5

 

Group / sex		Terminal Body weight (g)	Lungs and Bronchi weight (g)	Adjusted Lungs and Bronchi weight (g)
1M	Mean	286	1.151	1.126
	SD	18	0.128
	N	5	5
2M	Mean	280	1.162	1.164
	SD	10	0.081
	N	5	5
3M	Mean	287	1.270	1.239*
	SD	11	0.058
	N	5	5
4M	Mean	267	1.772	1.825 **
	SD	23	0.098
	N	5	5

 

	Control	Test substance
Dose group	1	2	3	4
Dose (µg/l)	0	5.86	31.3	157

 

M= Male F= female SD= Standard Deviation N= number of animals examined

** p < 0.01 * p< 0.0.5

 

7.5.2 /3 Macropathology results

Summary of findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Enlarged	0	0	1*	3	0	0	1*	5
Abnormal colour (pale)	0	0	1*	1	0	0	1*	4

Summary of findings in the mediastinal lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Enlarged	0	0	0	2	0	0	1*	5
Abnormal colour (pale)	0	0	0	1	0	0	1*	5

* No microscopic changes correlated with these findings

7.5.2 /4 Histopathology results

Lungs

 

Summary of treatment related findings in the lungs for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Alveolar Macrophages Increased, Minimal Slight Moderate	0 0 0	0 0 0	5 0 0	0 1 4	0 0 0	0 0 0	4 0 0	0 2 3
Total	0	0	5	5	0	0	4	5
Inflammation, Alveoli Slight	0	0	0	5	0	0	0	5
Total	0	0	0	5	0	0	0	5
Infiltrate, inflammatory cell, perivascular Minimal Slight	0 0	0 0	0 0	4 1	0 0	0 0	0 0	5 0
Total	0	0	0	5	0	0	0	5
Eosinophilic material- alveolar Slight Moderate	0 0	0 0	0 0	1 4	0 0	0 0	0 0	1 4
Total	0	0	0	5	0	0	0	5

 

Tracheobronchial lymph nodes

 

Summary of treatment related findings in the tracheobronchial lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	5	5	5	5	5	5	5	5
Increased Cellularity, paracortex Minimal Slight	0 0	0 0	0 0	2 0	0 0	0 0	0 0	3 1
Total	0	0	0	2	0	0	0	4

 

Mediastinal lymph nodes

 

Summary of treatment related findings in the mediastinal lymph nodes for animals killed after 4 weeks of treatment
Sex	Males				Females
Exposure level (µg/L)	0	5.86	31.3	157	0	5.86	31.3	157
Number of animals examined	0	0	0	2	0	0	1	5
Increased Cellularity, paracortex Minimal	0	0	0	1	0	0	0	5
Total	0	0	0	1	0	0	0	5

 

Conclusions:

The test article was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 5.86, 31.3 or 157 µg/L. Changes related to treatment with the test substance were seen in the lungs, tracheobronchial and mediastinal lymph nodes.
In the lungs these changes consisted of eosinophilic granular material within the alveoli and alveolar ducts of animals that received 157 µg/L; associated with this was the presence of increased numbers of macrophages and a neutrophilic infiltrate. Hyperplasia of the type II pneumocytes at the junction of the alveolar ducts and alveoli and perivascular mononuclear cell infiltrate was also evident. The combination of findings occurring in the lungs of animals that received 157 µg/L, together with lower body weight gain in that group, was such that the lung findings in this dose group were considered adverse.
Increased cellularity of the paracortex of the tracheobronchial and mediastinal lymph nodes were apparent, predominantly in animals that received 157 µg/L and 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.
On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 31.3 µg/L.

Executive summary:

The cumulative toxicity of the test item was assessed when administered to Wistar rats by snout-only inhalation administration for 6 hours per day, 5 days per week, over a period of 4 weeks. The study was designated to provide a rational basis for the assessment of the toxicological risk to man.

Three groups, each comprising five male and five female rats, received the test item at target exposure levels of 6, 30 or 150 µg/L. A similarly constituted control group received air at the same operating conditions as the 150 µg/L group. During the study, clinical condition, body weight, food consumption, haematology (peripheral blood), haematology (bone marrow), blood chemistry,organ weight, macropathology and histopathology investigations were undertaken.

The achieved gravimetric aerosol concentrations were 5.86, 31.3 and 157 µg/L (98, 104 and 105% of the target concentrations). The Mass Median Aerodynamic Diameters for all treated groups were within the ideal range (1-3µm) for a repeat dose inhalation study.

There were no test article-related deaths or effects on clinical signs, food consumption, haematology (bone marrow) or blood chemistry. Mean body weight gains at termination were lower for males and females exposed to 157 µg/L when compared with control (73% and 82% of control for males and females respectively). A similar effect was not apparent at the lower exposure levels.

Haematology showed lower group mean lymphocyte counts in both sexes exposed to 157 µg/L (0.66X and 0.64X control, respectively) and females exposed to 31.3 µg/L (0.77X control). Lower mean white blood cells counts were evident in both sexes exposed to 157 µg/L, reaching statistical significance in females (0.79X or 0.72X control for males and females respectively). Lower basophil and large unstained cell counts were also apparent; there were no effects in animals exposed to 31.3 or 5.86 µg/L.

Higher group mean lung and bronchi weights were observed for males and females exposed to 157 µg/L and males exposed to 31.3 µg/L; up to 1.62X control for males and 1.66X control for females (values adjusted for terminal body weight). There were no effects in animals exposed to 5.86 µg/L.

Macroscopic examination revealed enlargement of the tracheobronchial lymph nodes in the majority of animals exposed to 157 µg/L and one animal of each sex exposed to 31.3 µg/L. Enlarged mediastinal lymph nodes were apparent for two males and all females exposed to 157 µg/L and one female exposed to 31.3 µg/L. Lymph node enlargement was often accompanied by pallor. No microscopic findings associated with the enlarged lymph nodeswere seen in the two animals at 31.3 µg/L. There were no macroscopic effects in animals exposed to 5.86 µg/L.

Microscopically, changes related to treatment were seen in the lungs, tracheobronchial and mediastinal lymph nodes. In the lungs, changes related to treatment were present in the lungs of all animals that received 157 µg/L. Increased numbers of macrophages were present within the alveoli and alveolar ducts and were often hypertrophic with foamy cytoplasm and were accompanied by an infiltrate of neutrophils and type II pneumocyte hyperplasia at the bronchoalveolar junction. Eosinophilic material was present diffusely in the alveoli and alveolar ducts and had a granular appearance. An infiltrate of inflammatory cells consisting of mononuclear cells was present in predominantly the perivascular region but was also peribronchiolar in some cases. Treatment related changes were also seen in all the males and the majority of females that received 31.3 µg/L. The findings in the lungs were confined to a minimal macrophage response within the alveoli and alveolar ducts at 31.3 µg/L. These findings were considered to be a normal physiological response following inhalation of particle matter and were not adverse. The increase in macrophages was associated with the presence of eosinophilic granular material in the lungs (likely the test item) and was considered to be related to pulmonary clearance. There were no effects in animals exposed in 5.86 µg/L. In the tracheobronchial lymph nodes an increase in cellularity of the paracortex was evident in two males and four females exposed to 157 µg/L. An increase in cellularity of the paracortex was present in the mediastinal lymph nodes of one male and all females exposed to 157 µg/L.These findings 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 31.3 µg/L.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

31.3 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

28-day oral toxicity study complete and sufficient to fulfill the REACh annex VIII 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 two repeated-dose toxicity studies performed by the oral and inhalation routes:

Oral route:

The objective of this dose-range finding study was to select the dose-levels for a further OECD 421 reproductive/developmental toxicity screening.

Three groups of five male and five female Sprague-Dawley rats received the test item daily, by gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day for 14 days. A group of five males and five females received the vehicle alone, corn oil under the same experimental conditions and acted as a control group.

The animals were checked daily for mortality and clinical signs. Body weights were recorded once during the pre-treatment period, on the first day of treatment and then on days 4, 8, 11 and 14 before necropsy. Food consumption was recorded twice weekly during the treatment period.

On completion of the treatment period, animals were sacrificed. Kidneys, liver, heart, ovaries, spleen and testes were weighed and a full macroscopic post-mortem examination was performed.

 No unscheduled deaths related to the test item treatment occurred during the study. No clinical signs were observed in animals.

There were no test item-related changes in body weight and body weight gain during the study at any dose-level, and no relevant effects were recorded on food consumption.

No test item-related changes were observed in the mean organ weights or at the macroscopic post-mortem examination.

No adverse effects were observed in rats treated up to a dose-level of 1000 mg/kg/day for 14 days with the test item.

 

Inhalation:

The cumulative toxicity of the test item was assessed in a 4 -week inhalation toxicity study in rat. The study was performed in accordance with OECD guideline 412 and in compliance with Good Laboratory Practices.

The test item was administered by snout-only inhalation administration, for 6 hours a day, 5 days a week, for 4 weeks at achieved aerosol concentrations of 5.86, 31.3 and 157 µg/L.

A similarly constituted control group received air at the same operating conditions as the 157 µg/L group.

There were no test article-related deaths or effects on clinical signs, food consumption, bone marrow haematology or blood chemistry. Mean body weight gains at termination were lower for males and females exposed to 157 µg/L when compared with control.

Haematology showed lower group mean lymphocyte counts in both sexes exposed to 157 µg/L and to a lesser extent in females exposed to 31.3 µg/L. Lower mean white blood cells counts were evident in both sexes exposed to 157 µg/L.

Higher group mean lung and bronchi weights were observed for males and females exposed to 157 µg/L and to a lesser extent in males exposed to 31.3 µg/L. Macroscopic examination revealed enlargement of the tracheobronchial lymph nodes in the majority of animals exposed to 157 µg/L. Enlarged mediastinal lymph nodes were also apparent for two males and all females exposed to 157 µg/L.

Microscopically, changes related to treatment were seen in the lungs, tracheobronchial and mediastinal lymph nodes. In the lungs, these changes consisted of eosinophilic granular material within the alveoli and alveolar ducts of animals that received 157 µg/L, associated with this the presence of increased numbers of macrophages and a neutrophilic infiltrate. Hyperplasia of the type II pneumocytes at the junction of the alveolar ducts and alveoli and perivascular mononuclear cell infiltrate was also evident. The overall incidence and severity of the combination of findings occuring in the lungs of animals in conjunction with the lower body weight gain seen in these animals , was such that the lung findings at 157 µg/L were considered adverse. At 31.3 µg/L, the findings in the lungs were confined to a minimal macrophage response within the alveoli and alveolar ducts. These findings were considered to be a normal physiological response following inhalation of particle matter and were not adverse. The increase in macrophages was associated with the presence of eosinophilic granular material in the lungs which was likely the test item and was considered to be related to pulmonary clearance. Increased cellularity of the paracortex of the tracheobronchial and mediastinal lymph nodes were apparent, predominantly in animals that received 157 µg/L and 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.

On the basis of these findings, the No Observed Adverse Effect Level for this study was considered to be 31.3 µg/L.

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.