[1,2,4]Triazolo[1,5-c]pyrimidine, 2,2'-dithiobis[5-ethoxy-7-fluoro-

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2000 to 29 July 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

EC Number:

695-187-4

Cas Number:

166524-75-0

Molecular formula:

C14H12F2N8O2S2

IUPAC Name:

5-ethoxy-2-({5-ethoxy-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidin-2-yl}disulfanyl)-7-fluoro-[1,2,4]triazolo[1,5-c]pyrimidine

Test material form:

aerosol dispenser: not specified

Remarks:

migrated information: aerosol

Details on test material:

- Name of test material (as cited in study report): 2,2'-dithiobis (5-ethoxy-7-fluoro[1,2,4]triazolo[1,5C]pyrimidine) (DEDS)
- Appearance: lumpy yellow powder
- Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at receipt: 41 days
- Weight at study initiation: 160 - 225 g (males); 120 - 146 g (females)
- Fasting period before study: food was withheld during exposure and overnight prior to blood collection for clinical pathology evaluations
- Housing: individually in wire mesh cages suspended above cage board except during exposure periods
- Diet: Certified Rodent Lab Diet, ad libitum
- Water: reverse-osmosis treated (on-site) drinking water delivered by an automatic watering system ad libitum
- Acclimation period: 24 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 23.2 °C
- Humidity (%): 41.0 - 54.7 %
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark photoperiod

IN-LIFE DATES: From:24 October 2000 To: 23 February 2001

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

MMAD / GSD: 1.6 ± 2.10, 1.7 ± 2.44, and 1.9 ± 2.40 µm for the 20, 60 and 200 mg/m³ groups, respectively

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: four 8.6 L stainless steel conventional nose-only chambers with 20 exposure ports (one chamber was dedicated for each group during the study)
- Method of holding animals in test chamber: nose-only restraint tubes
- Source and rate of air: system ventilation air was provided from a humidified compressed air source. The exposure systems were operated at approximately atmospheric pressure
- System of generating particulates/aerosols: test atmospheres of test material were generated in the form of a dust aerosol. Aerosol atmospheres of test material were generated using jet mill air micronizers.
- Temperature and humidity in air chamber: exposure temperature: 20 - 22 °C; relative humidity: 33 - 44 %. The temperature and relative humidity were continuously monitored and recorded every 35 minutes during each exposure.
- Method of particle size determination: weekly measurements were recorded for the 20, 60 and 200 mg/m³ groups using an InTox cascade impactor.
- Treatment of exhaust air: exhaust from the exposure system was particulate filtered at the chamber prior to entering the in-house exhaust system, which provided HEPA filtration prior to discharge from the building

TEST ATMOSPHERE
- Brief description of analytical method used: actual exposure concentration within each chamber was measured by standard gravimetric methods. Four samples per day were collected from the 20, 60 and 200 mg/m³ groups. To confirm the absence of aerosol, the control chamber was sampled weekly, a total of four times during the exposure period.
- Animal exposure: for each daily exposure, the animals were loaded into nose-only exposure tubes, transported to the exposure room and exposed for the required duration (6 hours). Animals were rotated clockwise one position every two days and rotated by tier every six days in order to ensure that all animals in a given group received similar exposures over the course of the study.

VEHICLE
- Clean, filtered air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For each sample, the exposure atmosphere was drawn through a pre-weighed 25 mm glass fibre filter at a known flow rate for a known duration. Following sample collection, each filter was re-weighed and the concentration calculated as the filter weight difference divided by the sample volume.

Duration of treatment / exposure:

The exposure period was 6 hours per day.

Frequency of treatment:

Animals were exposed five days per week for four consecutive weeks.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 20, 60 and 200 mg/m³
Basis:
other: Target exposure levels

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 21 ± 1.3, 61 ± 3.3, 199 ± 10.9 mg/m³
Basis:
analytical conc.

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes

Details on study design:

- Pre-test period: prior to the initiation of the exposure period, each rat was acclimated to the nose-only restraint apparatus on an incremental basis. On consecutive days the animals were restrained for approximately 30 minutes, 1 hour and 3 hours, respectively.
- Rationale for animal assignment (if not random): animals were assigned using a computerised randomisation procedure
- Post-exposure recovery period: 5 males and 5 females per dose were euthanised and necropsied at the end of the exposure period (study week 4). The remaining five animals/sex/group were maintained for a 10 week non-exposure recovery period prior to necropsy (study week 14).

Examinations

Observations and examinations performed and frequency:

PRE-TEST EXAMINATIONS
On study week -2 the animals’ eyes were examined. On study weeks -1 and 0, individual body weights and detailed physical examinations were recorded. Individual food consumption was recorded for study week -1.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed twice daily, once in the morning and once in the afternoon, for mortality and moribundity. During the exposure period, the animals were observed every hour for mortality and moribundity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: clinical examinations were conducted on all animals prior to exposure at the time of loading into the nose-only restraint tubes. On non-exposure days and during the recovery period, clinical examinations were performed once daily in the morning. Detailed physical examinations were conducted weekly, beginning one week prior to test material administration and just prior to necropsy. Clinical examinations were not performed on days when detailed physical examinations were conducted.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded weekly, beginning two weeks prior to exposure.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly, beginning one week prior to exposure. Food intake was calculated as g/animal/day for the corresponding bodyweight intervals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: study week -2 and study week 4
- Dose groups that were examined: all animals in all dose groups were examined using an indirect ophthalmoscope and a slit lamp biomicroscope, preceded by mydriasis.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: all rats (study week 4) and all male rats (study week 14). Blood was taken from fasted animals via the vena cava at necropsy.
- Animals fasted: Yes (overnight).
- Parameters checked included: total leukocyte count*, erythrocyte count*, haemoglobin*, haematocrit*, mean corpuscular volume*, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration*, platelet count*, prothrombin time, reticulocyte count, activated partial thromboplastin time and differential leukocyte count* (neutrophil, lymphocyte, monocyte, eosinophil, basophil)
* Performed on males only at the recovery necropsy

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: all rats (study week 4) and all male rats (study week 14). Blood was taken from fasted animals via the vena cava at necropsy.
- Animals fasted: Yes (overnight).
- Parameters checked included: albumin*, total protein, globulin, albumin/globulin ratio, total bilirubin, urea nitrogen, creatinine, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, glucose*, total cholesterol, calcium*, chloride*, phosphorus*, potassium* and sodium*
* Performed on males only at the recovery necropsy

URINALYSIS: Yes
- Time schedule for collection of urine: study week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (overnight).
- Parameters checked included: specific gravity, pH, urobilinogen, total volume, colour, appearance, protein, glucose, ketones, bilirubin, occult blood, leukocytes, nitrites and microscopy of sediment.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
A complete necropsy was conducted on all animals. All animals were euthanised at the scheduled necropsies by isoflurane anaesthesia followed by exsanguination. The necropsy included, but was not limited to, examination of the external surface, all orifices and the cranial, thoracic, abdominal and pelvic cavities including viscera. At the time of necropsy the following tissues and organs were collected and preserved in 10 % neutral buffered formalin:
adrenals, aorta, bone (femur with joint), bone with marrow (sternebrae), bone marrow smear (from femur)*, brain (forebrain, midbrain, hindbrain), cervix, coagulating glands, epididymides, eyes with optic nerve, exorbital lacrimal glands, gastrointestinal tract (oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum), Harderian glands, heart, kidneys, larynx, liver (sections of three lobes), lungs (including bronchi, fixed by inflation with fixative), lymph node (mesenteric, mediastinal), mammary gland (females only), nasal and oral tissues#, ovaries with oviducts, pancreas, parathyroids, peripheral nerve (tibial), pituitary, prostate, salivary glands (mandibular), seminal vesicles, skeletal muscle (rectus femoris), skin, spinal cord (cervical, midthoracic, lumbar), spleen, testes, thymus, thyroids, trachea, urinary bladder, uterus with vagina and gross lesions (when possible).
* not placed in formalin; to be examined only if scientifically warranted
# the entire head was removed and preserved

ORGAN WEIGHTS
The following organs were weighed from all animals at the primary necropsy: adrenals, brain, kidneys, liver, lungs (prior to inflation with fixative), ovaries, spleen, testes and Epididymides and thymus.
The brain, kidneys, liver and lungs were weighed from all animals at the recovery necropsy. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
Histological processing and microscopic examination was performed on all tissues (listed in the gross pathology section above) by the sponsor.

Statistics:

STATISTICAL METHODS (SPONSOR)
Body weight data were evaluated using repeated measures analysis of variance for time, sex and dose. If significant (alpha = 0.02), the analysis was repeated separately for each sex without examining the results of other factors. The time-dose interaction was examined next. If the time-dose interaction was statistically significant (alpha = 0.5), linear contrasts tested the time-dose interaction for the comparisons of each dose group to the control group. A Boneferroni correction was applied to the alpha level to compensate for the multiple comparisons with the control group. The corrected comparison-wise error rate of alpha = 0.02 was reported so direct comparison could be made to the Pillai's Trace p-values generated. Body weights at the recovery time points were evaluated using a two-way analysis of variance with the factors of sex and dose. For these parameters, the first examination was whether the sex-dose interaction was significant (alpha = 0.05); if it was, a one way analysis of variance was done separately for each sex. Comparisons of individual dose groups to the control group were made with the Dunnett's test only when a statistically significant dose effect existed at alpha = 0.05.

STATISTICAL METHODS (TEST FACILITY)
Statistical tests were conducted using two-tailed tests for minimum significance levels of 5 and 1 % comparing the test material-exposed groups to the air control group by sex. Statistical analyses were not performed of the number of animals was two or less. Food consumption, clinical pathology data and organ weights were subjected to a one-way analysis of variance, followed by Dunnett's test if the analysis of variance revealed statistical significance (p<0.05). Clinical laboratory values for leukocytes that occur at low incidence were not subjected to statistical analysis.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Possible test material-related effect - see "Details on results" for information

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Possible test material-related effect - see "Details on results" for information

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Possible test material-related effect - see "Details on results" for information

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See "Details on results" for information

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
All animals survived to the scheduled necropsies. There were no test material-related clinical observations. Those findings that were observed were limited to single animals, were not observed with a dose-related incidence and/or were common findings in laboratory rats of this age and strain.

BODY WEIGHT AND WEIGHT GAIN
There were no test material-related effects on body weights or body weight gains. Analysis of body weight data revealed a statistical significance at p<0.05 when evaluating the time-dose interaction at all dose levels combined. Each dose group was statistically significant at p<0.02 when analysed individually compared to the control group. Mean body weights in the 200 mg/m³ group males and females were lower than the control group during the recovery period (study weeks 5 to 14). However, as these changes only occurred during the recovery period, a correlation to test material administration could not be made.

FOOD CONSUMPTION
There were no test material-related effects on food consumption. Compared to the control group males, the 60 mg/m³ group males had a statistically significant (p<0.05) decrease in food consumption for study weeks 12 - 13. However, the difference was slight (2 grams), occurred during the recovery period and was not considered the result of exposure to test material.

OPHTHALMOSCOPIC EXAMINATION
There were no test material-related ophthalmic lesions. All findings observed were typical in prevalence and appearance for laboratory rats of this age and strain.

HAEMATOLOGY
Mean white blood cell count and absolute lymphocyte count were lower in the 200 mg/m³ group males at the study week 4 evaluation (29 and 28 % lower than the control group means, respectively). This small lymphocyte-based change was considered possibly test material-related, but is of uncertain toxicological significance. Similar changes were not observed in the females or in males following the recovery period when compared to the control group. No other test material-related differences were noted.
There was, however, a statistically significant (p<0.05) decrease in mean haemoglobin levels in the 60 and 200 mg/m³ group males at the study week 4 evaluation. This difference was not considered to be test material-related since the magnitude of the difference from the control group was small, it was not dose-related and all other red blood cell-dependent parameters were unaffected.

CLINICAL CHEMISTRY
Possible test material-related effects on serum chemistry parameters were noted at the primary necropsy and consisted of lower mean calcium and phosphorus levels in the 20, 60 and 200 mg/m³ group males. These differences were statistically significant (p<0.05 or p<0.01) when compared to the control group. However, since the differences from the control group were small and/or there were no clear dose-related patterns, no toxicological significance was attributed to these changes from the control group. There were no other test material-related effects in serum chemistry parameters.
However, there were several statistically significant (p<0.05 or p<0.01) differences when the treated groups were compared to the control group. Mean albumin in the 200 mg/m³ group males was lower compared to the control group at the primary necropsy. The difference was not considered to be test material-related since its magnitude was small and there was no evidence of a dose relationship. Mean glucose levels were lower in the 20 mg/m³ group males, and mean creatinine and potassium levels were lower in the 20 and 60 mg/m³ group males at the time of primary necropsy. Since these differences were not noted in the 200 mg/m³ group males, they were not considered test material-related effects. In addition, the differences for potassium resulted from high and spurious values for individual control males. There were no test material-related effects at the recovery necropsy.

URINALYSIS
Urinalysis parameters were unaffected by test material exposure.

ORGAN WEIGHTS
A possible test material-related effect on organ weight was noted at the primary necropsy and consisted of increased lung weights in the 20, 60 and 200 mg/m³ group males and females. At the primary necropsy, mean absolute lung weights were significantly (p<0.01) increased in the 60 and 200 mg/m³ group males and females and mean relative lung weights were significantly (p<0.01) increased in the 20, 60 and 200 mg/m³ group males and females compared to control group means. Although not statistically significant, similar increases in mean absolute lung weights were observed in the 20 mg/m³ group males and females.
There were no other test material-related changes in organ weights at the primary necropsy. However, there were a few statistically significant (p<0.05) differences when the control and treated groups were compared. Mean relative liver weight was lower in the 200 mg/m³ group males and relative kidney and brain weights were lower in the 60 and 200 mg/m³ group females, respectively. Since the differences from the control group means were small in magnitude, they were not considered test material-related. There were no test material-related effects on organ weights at the recovery necropsy.

GROSS PATHOLOGY
There were no test material-related macroscopic findings at the primary or recovery necropsies. Those findings observed were limited to single animals in various groups (including the control group) and/or were findings commonly observed in laboratory rats.

HISTOPATHOLOGY
At the primary necropsy, histopathologic lesions attributable to inhalation of the test material were restricted to the respiratory tract, specifically lungs and nasal tissues, and the associated mediastinal lymph nodes and were found for rats exposed to all levels of test material.

Lungs of exposed rats had a mild centriacinar inflammatory reaction involving the terminal bronchioles, alveolar ducts, and adjacent alveoli that affected all lung lobes uniformly. The reaction was characterised by accumulations of vacuolated macrophages and occasional neutrophils in the lumen at this site. There was very slight thickening of the walls of the alveolar ducts and terminal bronchioles by these inflammatory cells and low numbers of other mononuclear inflammatory cells and connective tissue cells.
Particulate material was not readily discernible in the macrophages. Rats exposed to 20 mg/m³ had very slight inflammation; the response increased with concentration, with the reaction graded as slight for rats exposed to 60 mg/m³ and moderate for rats exposed to 200 mg/m³. The response was similar between the sexes, although males had an equivocally greater response than females at 60 mg/m³. These changes correlated with the elevated absolute and/or relative lung weights observed in the test material-treated groups.
The lesion in nasal tissues was termed hyperplasia, goblet cell, and was of minimal degree. In affected sites, most clearly noted along the nasal septum in the first and second nasal sections and the pharyngeal dust in the third and fourth sections, the proportion of goblet cells was increased. In these sites the mucous appeared thicker due to a multilayered appearance of mucin vacuoles and the nuclei of goblet cells were pseudostratified. Affected mucosa had a slightly irregular surface and a low number of gland-like collections of goblet cells were found in some affected sites. The lesions were not associated with necrosis, ulceration nor inflammation and were likely an adaptive or protective response rather than a truly adverse effect.
Rats from all dose groups, including controls, tended to have focal goblet cell hyperplasia that was restricted to the mucosa of the first nasal section, primarily on the nasal septum. Focal goblet cell hyperplasia was considered a spontaneous change and was found in 2 or 3 controls or rats exposed to 20 or 60 mg/m³ test material. However, all males and three of five female rats exposed to 200 mg/m³ had more extensive goblet cell hyperplasia that was considered related to treatment.
There was diffuse hyperplasia of the mediastinal lymph nodes in many of the exposed rats. This was graded as slight or moderate and affected all rats exposed to 200 mg/m³ and 2 males exposed to either 20 or 60 mg/m³ and 3 females exposed to 60 mg/m³. As these lymph nodes drain the lung, this effect was considered secondary to the pulmonary effects.

Effect levels

Dose descriptor:

NOAEL

Effect level:

< 20 mg/m³ air

Based on:

test mat.

Sex:

male/female

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1: Treatment-related Histopathologic Lesions after Four-weeks of Exposure

Organ/ observation	Exposure level (mg/m³)
	Male				Female
	0	20	60	200	0	20	60	200
Lungs
Inflammation, subchronic, bronchioloalveolar, multifocal
Very slight	0	5	0	0	0	5	3	0
Slight	0	0	5	2	0	0	2	3
Moderate	0	0	0	3	0	0	0	2
Nasal Tissue
Hyperplasia, goblet cell, respiratory mucosa
Focal, unilateral, very slight	0	2	1	0	0	2	0	1
Focal, bilateral, very slight	2	0	1	0	2	0	1	0
Slight	1	0	0	0	0	0	0	0
Diffuse, bilateral, very slight	0	0	1	4	0	0	1	3
Slight	0	1	0	1	0	0	0	0
Lymph Node - Mediastinal
Hyperplasia, diffuse
Slight	0	1	2	3	0	0	3	1
Moderate	0	1	0	2	0	0	0	4

Bold digitsindicate effect attributed to treatment

 

Table 2: Histopathologic Observations after Ten weeks of Recovery

Organ/ observation	Exposure level (mg/m³)
	Male				Female
	0	20	60	200	0	20	60	200
Lungs
Inflammation, subchronic, bronchioloalveolar, multifocal
Any grade	0	0	0	0	0	0	0	0
Nasal Tissue
Hyperplasia, goblet cell, respiratory mucosa
Focal, unilateral, very slight	1	1	2	0	2	0	0	0
Focal, bilateral, very slight	0	3	0	0	1	0	0	0
Slight	1	0	0	0	0	0	1	0
Multifocal, bilateral, very slight	0	0	1	0	0	0	0	0
Lymph Node - Mediastinal
Hyperplasia, diffuse
Slight	0	1	3	0	1	0	1	1

 

Table 3: Summary of Organ Weight Means (grams) - Animals Necropsied on Study Week 4

Organ	Male
	0 mg/m³	20 mg/m³	60 mg/m³	200 mg/m³
Brain	1.75	1.72	1.72	1.72
Liver	5.54	4.92	5.17	4.89
Kidneys	1.54	1.39	1.46	1.49
Spleen	0.44	0.37	0.41	0.38
Lungs	0.98	1.06	1.19**	1.30**
Epididymides	0.52	0.51	0.58	0.52
Testes	2.44	2.34	2.48	2.31
Thymus gland	0.2044	0.1571	0.1763	0.1660
Adrenal glands	0.0535	0.0494	0.0532	0.0522
Organ	Female
	0 mg/m³	20 mg/m³	60 mg/m³	200 mg/m³
Brain	1.66	1.61	1.67	1.65
Liver	3.64	3.68	3.68	3.90
Kidneys	1.14	1.11	1.07	1.14
Spleen	0.33	0.34	0.34	0.33
Lungs	0.78	0.88	1.00**	1.15**
Ovaries	0.0535	0.0508	0.0538	0.0601
Thymus gland	0.1709	0.1808	0.1890	0.1809
Adrenal glands	0.0574	0.0586	0.0551	0.0613

** Significantly different from the control group at 0.01 using Dunnett's test

 

Table 4: Summary of Organ Weight Means (grams) - Animals Necropsied on Study Week 14

Organ	Male
	0 mg/m³	20 mg/m³	60 mg/m³	200 mg/m³
Brain	1.88	1.90	1.88	1.89
Liver	7.85	8.25	6.93	7.47
Kidneys	1.95	1.99	1.86	1.88
Lungs	1.26	1.31	1.19	1.20
Organ	Female
	0 mg/m³	20 mg/m³	60 mg/m³	200 mg/m³
Brain	1.78	1.78	1.74	1.76
Liver	5.19	4.96	4.83	5.12
Kidneys	1.33	1.33	1.28	1.34
Lungs	0.97	0.97	1.03	1.06

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the No Observed Adverse Effect Level (NOAEL) of the test material was determined to be less than 20 mg/m³ when male and female rats were exposed to the test material as a dust aerosol for four consecutive weeks.

Executive summary:

The repeated dose (inhalation) toxicity of the test material was investigated in a study which was conducted under GLP and conditions and in accordance with the standardised guidelines OECD 412 and EU Method B.8.

During the study, groups of 10 male and 10 female Fischer 344 rats were exposed to 0 (filtered air), 20, 60 or 200 mg/m³ test material, in nose-only chambers for 6 hours per day, 5 days per week for 4 consecutive weeks. At the end of the exposure period, 5 males and 5 females per group were euthanised and necropsied. The remaining 5 animals per sex and group were maintained for a 10 week non-exposure recovery period prior to necropsy on study week 14. During the study, the animals were observed for mortality and clinical signs. Food consumption was monitored and ophthalmic and clinical pathology examinations were undertaken. At necropsy animals were subjected to a gross pathologic examination; organ weights were recorded and histologic processing and microscopic examination of tissues was undertaken.

All animals survived to the scheduled necropsies. There were no test material-related clinical observations or effects on body weight or food consumption data. There were no test material-related effects on haematology, serum chemistry or urinalysis parameters for females or for urinalysis parameters for males. There were no ophthalmic lesions attributed to the test material. There were no test material-related macroscopic findings at the scheduled necropsies.

For the 200 mg/m³ group males at the primary necropsy, mean white blood cell counts and mean absolute lymphocyte counts were 28 - 29 % lower than respective control values. These possible test material-related differences were of uncertain toxicological significance. No differences were observed following the recovery period. Possible test material-related effects in serum chemistry parameters were noted at the primary necropsy and consisted of lower mean calcium and phosphorus levels in the 20, 60 and 200 mg/m³ group males. The differences from the control group were not considered toxicologically significant, since they were small in magnitude and/or clear dose-related patterns were not present. Serum chemistry parameters in the exposed groups at the recovery necropsy were similar to control values. Test material-related effects on organ weights at the primary necropsy consisted only of increased lung weights in the 20, 60 and 200 mg/m³ group males and females. There were no test material-related effects on organ weights at the recovery necropsy.

Test material-related histopathologic findings were observed in the lungs, nasal tissues and local (mediastinal) lymph nodes in the 20, 60 and 200 mg/m³ group males and females. In the lungs, inflammation was localised to the terminal bronchiole/alveolar dust junction (i.e., bronchioloalveolar junction) in all 20, 60 and 200 mg/m³ group males and females.The degree of inflammation involved increased with the level of exposure. These changes correlated with elevated absolute and/or relative lung weights observed for the test material-treated groups.

In the nasal tissues, goblet cell hyperplasia was present in the respiratory mucosa at all anatomic sites examined in rats exposed to 200 mg/m³. Goblet cell hyperplasia was not associated with necrosis, ulceration nor inflammation and was likely an adaptive or protective response. In the lymph nodes, mediastinal hyperplasia was considered a secondary response in two males and three females in the 60 mg/m³ group and all rats in the 200 mg/m³ group. At the recovery necropsy, all treatment-related effects had resolved.

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) of the test material was determined to be less than 20 mg/m³ when male and female rats were exposed to the test material as a dust aerosol for four consecutive weeks.