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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
aerosol dispenser: not specified
Remarks:
migrated information: aerosol
Details on test material:
Name of test substance: Trilon BD, Ethylendiamintetraessigsäure, Dinatriumsalz; Na2H2EDTA; Disodium dihydrogen ethylenediaminetetraacetate
Test substance No.: 09/0628-3
Batch Identification: Lot 64580436W0
CAS No.: 139-33-3
Purity: 88.1 g/100 g
Homogeneity: Homogeneous
Storage stability: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: about 7 weeks
- Housing: up to 5 animals per cage in Polysulfon cages (H-Temp [PSU])
- Diet (e.g. ad libitum): 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switzerland) ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): A light/dark rhythm of 12 hours was maintained: 06.00 a.m. 06.00 p.m. light, 06.00 p.m. 06.00 a.m. dark

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: Test group 1 (0.5 mg/m3): MMAD (µm) 2.3-2.8; Geometric standard deviation 1.7-2.2
Test group 2 (3 mg/m3): MMAD (µm) 2.0-2.4; Geometric standard deviation 1.8-2.0
Test group 3 (15 mg(m3): MMAD (µm) 2.3-2.5; Geometric standard deviation 1.8-2.1
Details on inhalation exposure:
For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned dilution air and passed into the inhalation system. To achieve stable concentration in the atmosphere, a part of generated atmosphere was replaced by fresh conditioned air.
The inhalation atmosphere was maintained inside aerodynamic exposure systems, consisting of a cylindrical inhalation chamber made of stainless
steel sheeting and cone-shaped outlets and inlets. The rats were restrained in glass exposure tubes. Their snouts projected into the inhalation chamber and thus they inhaled the aerosol. The exposure systems were located in exhaust hoods in an air conditioned room. All test groups were exposed for 6 hours on each workday over a time period suitable to reach 65 exposures. The animals did not have access to water or feed during the exposure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres were analyzed by gravimetry in all test groups including. This analytical method is judged to be valid because the test substance does not possess an appreciable vapor pressure. Daily means were calculated based on 2 measured samples in test group 1 and 3 measured samples per concentration and exposure in test groups 2 and 3. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.
Scattered light photometry was used to continuously monitor the constancy of concentrations of test substance aerosols in the inhalation systems. To this end the inhalation atmosphere was continuously sampled by the measuring devices.
The particle size analysis was carried out with a cascade impactor.


Duration of treatment / exposure:
Exposures: 6 hours per day

Frequency of treatment:
daily (5 consecutive days/week), 13 weeks, 65 exposures in total
Doses / concentrations
Remarks:
Doses / Concentrations:
0.5, 3, 15 mg/m3
Basis:
nominal conc.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were examined for evident signs of toxicity or mortality twice a day on working days and once a day on Saturdays, Sundays and public holidays

CLINICAL OBSERVATIONS: Yes
The clinical condition of the test animals was recorded once during the pre-exposure period and on post-exposure observation days and at least 3 times (before, during and after exposure) on exposure days. During exposure only a group wise examination was possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: days 0, 42, 84

BODY WEIGHT: Yes
- Time schedule for examinations: at start of the pre-exposure, at start of the exposure, then twice a week (Monday, Friday), and prior to gross necropsy

FOOD CONSUMPTION:
- Food consumption was determined cage-wise weekly (Monday-Friday) and calculated as g food/animal/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before the start of the exposure period (day -1/ -2) the eyes of all animals, and at the end of the study (day 82/83) the eyes of the animals of test group 0 (control group) and test group 3 (high concentration) were examined for any changes in the refracting media with an ophthalmoscope after administration of a mydriatic.

HAEMATOLOGY: Yes
All animals per test group and sex at the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
Parameters: WBC, RBC, HGB, HCT, MCV, MCH, MCHC, PLT, DBC, RET

CLINICAL CHEMISTRY: Yes
All animals per test group and sex at the end of the administration period
- Animals fasted: Yes
Parameters. ALT, AST, ALP, GGT, NA, K, CL, INP, CA, UREA, CREA, GLUC, TBIL, TPROT, ALB, GLOB, TRIG, CHOL

URINALYSIS: Yes
All animals per test group and sex at the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity / grip strength / motor activity / Open filed observations

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals assigned for light microscopic examination were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. The exsanguinated animals were necropsied and assessed by gross pathology.

Organ weights: all animals sacrificed on schedule
Anesthetized animals, Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Lung, Ovaries, Spleen, Testes, Thymus, Thyroid glands, Uterus

Organ/Tissue Fixation:
The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution:
All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain with olfactory bulb, Cecum, Colon, Duodenum, Epididymides, Esophagus, Extraorbital lacrimal gland, Eyes with optic nerve and eyelid (modified Davidson’s solution), Femur with knee joint, Harderian glands, Heart, Ileum, Jejunum, Kidneys, Larynx, Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric lymph nodes), Mammary gland (male + female), Nose (nasal cavity), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Tongue, Trachea, Ureter, Urethra, Urinary bladder, Uterus

HISTOPATHOLOGY: Yes
List of organs and tissues of histological examinations: All gross lesions, Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Colon, Duodenum, Esophagus, Femur with knee joint, Heart, Ileum, Jejunum, Kidneys, Larynx (3 levels), Liver, Lung, Lymph nodes (tracheobronchial, mediastinal and mesenteric), Mammary gland (female), Nasal cavity (4 levels), Ovaries, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate, Rectum, Salivary glands (mandibular and sublingual glands), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Teeth, Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus
Statistics:
Body weight, body weight change - comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the
hypothesis of equal means
Feces, rearing, grip, strength length, forelimbs, grip, strength length, hindlimbs, footsplay, test, motor activity - Non-parametric one-way analysis
using KRUSKAL-WALLIS test (twosided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two-sided) for the equal medians
Blood parameters - For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) with Bonferroni-Holm adjustment for the hypothesis of equal medians
Weight parameters - Non-parametric one-way analysis using KRUSKAL-WALLIS test (twosided).If the resulting p-value was equal or less than 0.05, a pairwise
comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.
Mortality:
no mortality observed
Description (incidence):
One male rat (No. 33) of the high concentration group showed discoloration (orange) of feces and smeared fur (red) in anogenital region. The effects were observed in the morning before exposure on study day one. Therefore, the animal was sacrificed after the first exposure on study day one. As no other animals of this group showed similar clinical signs of toxicity during the whole exposure period of 90 days (65 exposures), the findings in animal No. 33 were considered to be incidental and not substance-related. No further deaths were recorded in the study.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see details on results
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results

Effect levels

open allclose all
Dose descriptor:
LOAEC
Effect level:
15 mg/m³ air
Based on:
test mat.
Sex:
female
Basis for effect level:
other: histopathology
Dose descriptor:
NOAEC
Effect level:
> 15 mg/m³ air
Based on:
test mat.
Sex:
male
Basis for effect level:
other: histopathology
Dose descriptor:
NOAEC
Effect level:
3 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Organ weights:

Relative changes of absolute liver and lung weights in comparison to the control

 

Male animals

Female animals

Test group

(mg/m³)

1

(0.5)       

2

(3)

3

(15)

1

(0.5)       

2

(3)

3

(15)

Liver

 

 

 

107% 

102%

109%*

Lungs

99%

93%

108%*

107%*

112%**

124%**

* : p <= 0.05, **: p <= 0.01

 

All other mean absolute weight parameters did not show significant differences when

compared to the control group 0.

Relative organ weights

When compared with control group 0 (=100%), the following mean relative organ weights

were significantly changed (statistically significant changes printed in bold):

 

Female animals 

Test group

(mg/m³)

1

(0.5)        

2

(3)

3

(15)

Lungs

104%

112%**

120%**

* : p <= 0.05, **: p <= 0.01

 

All other mean relative weight parameters did not show significant differences when compared to the control group 0.

The increased lung weights in males of test group 3 (15 mg/m³) and females of all test groups might be related to the treatment. No histopathologic finding could explain the weight increase. In males, the relative lung weight was not significantly changed. Furthermore was the lung weight of female animals within the range of historical control data. Therefore the lung weight increase was regarded to be not adverse.

Treatment-related findings were observed in male and females with incidences and grading according to the table below

 

Male animals

Female animals

Test group

(mg/m³)

0

(0)

1

(0.5)

2

(3)

3

(15)

0

(0)

1

(0.5)

2

(3)

3

(15)

No.of animals

10

10

10

10

10

10

10

10

Hyperplasia (m)focal

0

0

0

0

0

0

0

1

Metaplasia, squamous

0

0

1

6

0

0

1

3

·                  Grade1

 

 

1

2

 

 

1

1

·                  Grade2

 

 

 

4

 

 

 

2

Epithelial alteration

2

4

6

4

1

7

8

6

·                  Grade1

2

4

5

1

1

7

8

2

·                  Grade2

 

 

1

3

 

 

 

4

Infiltrates, granulocytic

 

 

 

 

 

 

 

2

·                 Grade2

 

 

 

 

 

 

 

2

Whenever no grading was given the finding was recorded to be present

Animals of all test groups as well as single control animals revealed minimal to slight epithelial alteration at the base of the epiglottis. The term was used, if at the base of the epiglottis a small focal area was covered by flattened epithelium, which differed from the normal cuboidal to columnar laryngeal epithelium. Secondary, some animals of test group 3 (15 mg/m³) and 2 (3 mg/m³) showed a minimal to slight focal squamous metaplasia in this region. These findings were regarded to be test substance related and adaptive. One female of test group 3 (15 mg/m³) revealed a small focal hyperplasia of the laryngeal epithelium at the base of the epiglottis and in addition slight granulocytic cell infiltrates. These infiltrates were also observed in a second female of this test group. These findings were regarded to be treatment related and adverse.

Microscopic findings in lung and their grading

 

Maleanimals

Femaleanimals

Test group

(mg/m³)

0

(0)

1

(0.5)

2

(3)

3

(15)

0

(0)

1

(0.5)

2

(3)

3

(15)

No.ofanimals

10

10

10

10

10

10

10

10

Histiocytosisalveolar,d

0

0

6

10

0

0

5

10

·                  Grade1

 

 

6

4

 

 

5

6

·                  Grade2

 

 

 

6

 

 

 

4

Metaplasiamucouscells

0

0

0

4

0

0

0

5

·                  Grade1

 

 

 

4

 

 

 

5

Animals of test group 2 and 3 (3 and 15 mg/m³) revealed minimal to slight increased numbers of alveolar histiocytes. These histiocytes showed an eosinophilic cytoplasm, occasionally with clear vacuoles and were located as single cells within the alveoli all over the lung lobes. When compared to the control animals, males and females of test group 3 (15 mg/m³) showed a minimal to slight increase in number of mucous cells in the large bronchi. These findings were regarded to be treatment related effects and adaptive.

Testes

Tubular degeneration (up to moderate) was observed in control (8 animals affected) and test group 3 animals (7 animals affected) in similar incidences. This finding was characterized by randomly affected (not stage specific) tubules with sloughed spermatogenic cells, vacuolation of the spermatogenic epithelium or missing germ cell layers. This effect in the testis is likely due to the technical exposure scenario (heat [e.g. Brock WJ et al., 1996] and possibly evading movements by the animals leading to pressing backwards in tubes), rather than being a direct effect of the test substance as this finding is also found in similar incidences in control animals.

All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Applicant's summary and conclusion

Executive summary:

Inhalation exposure of rats to Trilon BD for 90 day (65 exposures) did not lead to any substance-related clinical signs of toxicity. Nor were there any effect in clinical chemistry, hematology. Histological examination revealed some effects in larynx at the highest tested concentration of 15 mg/m³ in female animals. No signs of systemic toxicity were observed up to a concentration of 15 mg/m³. Signs of local toxicity were observed only at the high concentration of 15 mg/m³ in female animals.

Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effects in larynx was 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.