Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

One 28-day oral gavage study in rats using pentapotassium DTPA and one 28-day oral, drinking water study using pentasodium DTPA. Additional information comes from published literature on othe salts of DTPA such as zinc and calcium
Subchronic 90-day OECD 413 guideline compliant inhalation study performed on suitable read-across substance, Na2H2EDTA.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
75 mg/kg bw/day

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-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: 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
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
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
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
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
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
Critical effects observed:
not specified

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.

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
3 mg/m³
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
2013-2014
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-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: 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
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
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
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
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
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
Critical effects observed:
not specified

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.

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
3 mg/m³
Study duration:
subchronic
Species:
rat

Additional information

Oral studies

There are two standard guideline repeated dose toxicity studies available for DTPA. The studies were conducted using either the potassium or sodium salt. Overall, the two studies are consistent in the adverse effects identified, particularly with respect to the apparent target organs and the no effect levels. The only significant difference between the two studies is the mortality encountered in the study using gavage administration. This increased mortality in the males (4/5) and females (1/5) was probably due to a bolus dose effect rather than a greater toxicity of the potassium salt since the sodium DTPA was administered via the drinking water rather than gavage. With the exception of the high dose group mortality, and the decreased body weight and food consumption in the high and mid dose groups the other adverse effects identified were relatively minimal (changes in clinical chemistry parameters and some alterations in urine parameters (high dose only)). The liver appears to have been a target organ for toxicity, but these effects may have been due to the decreased bodyweight and stress rather than direct compound related toxicity.

The relatively minimal systemic toxicity observed is also consistent with the low degree of absorption following oral administration and its subsequent rapid excretion (half life of approx 2 hours). DTPA is chemically stable and not reactive or metabolized, thus its toxicity is generally associated with the ability to chelate essential metals. DTPA is known to be capable of producing deficiencies in zinc when administered systemically or orally. The effects in the 2 repeated dose studies in the high dose groups are consistent with the development of a nutritional deficiency, such as a zinc deficiency. The reduction in food intake and associated bodyweight decrease are known to be associated with deficiencies of zinc, as the animals reduce their food intake in an effort to trigger catabolism of their tissues to release more zinc. If these studies had continued for a longer period then it is highly likely that more obvious changes in pathology consistent with a zinc deficiency would appear.

Further support for the toxicity of DTPA being linked to its ability to remove essential metals such as zinc comes from toxicity studies conducted using the zinc salt of DTPA. In a number of comparative studies (predominantly studying developmental toxicity), the zinc salt of DTPA has been significantly less toxic compared to the calcium salt of DTPA.

Due to the nature of DTPA toxicity, it is unlikely that a longer study would identify additional adverse effects or a significantly lower no effect level. There is a finite amount of essential metals in the diet and body. DTPA can only bind to metals during the brief period when it comes into contact (either in the gut or the blood). If a dose of DTPA is insufficient to significantly impact an animal’s intake of essential metals then increasing the number of days of exposure will not have a more severe effect, i.e. there will be a threshold. Thus, rather than leading to a significantly lower no effect level, a longer term study would probably just lead to a greater degree of zinc deficiency thus it would show an increased severity of the effects observed in the shorter study.

There are no longer term guideline studies for DTPA, however there is a longer term study available for another salt of DTPA. In this study Calcium DTPA (approximately 44 mg/DTPA/kg bw) was administered via intraperitoneal injection twice per week to rats for 44 weeks. There were no observed effects on any of the parameters examined (including body weight, clinical signs, pathology, urinalysis, clinical chemistry and hematology). However, by administering DTPA only 2 times per week, there was a recovery period between the doses where the rats would have been able to replace essential nutrients like zinc. Therefore, whilst this study does not indicate any adverse effects following long term exposure to DTPA, if the dose had been administered daily there would probably have been toxicity since it would be equivalent to a daily oral dose of approximately 400 and 800 mg/kg bw oral dose (assuming 5 - 10% absorption in the gut). This study does however illustrate that the toxicity of DTPA is reversible, since there was apparently sufficient time within this study design for the animals to recoup lost essential elements.

Based on the available data it is therefore argued that no further sub-chronic or chronic testing is necessary. Rather it is proposed that the DNELS can be calculated using the available 28-day studies. As such the starting point for the calculation of the DNELs is taken from drinking water study conducted using pentasodium DTPA. In this study the NOEL was 75 mg/kg bw.

Inhalation studies:

A sub acute inhalation study was performed using the chelating agent Disodium EDTA. In this study disodium EDTA was demonstrated to produce effects consistent with irritation in the respiratory tract including the larynx and lungs at all doses tested. At the highest dose tested (1000 mg/m3), 6 of the animals (out of 20 male rats) died either on the day of first exposure or the day afterwards. Cause of death was attributed to congestion and edema and multifocal hemorrhages in the lungs. There were no mortalities in the lower doses (300 and 30 mg/m3). Aside from the irritation of the respiratory tract there were no other treatment related effects. The effects noted in the respiratory tract were considered to be local and not the result of some systemic toxicity.

In a subchronic repeated dose toxicity study (BASF, 2014) according to OECD guideline No. 413 Wistar rats were exposed to a respirable dust aerosol of Na2H2EDTA for 6h/d on 5 consecutive d/w for 13 weeks (65 exposures in total) at concentrations of 0, 0.5, 3 or 15 mg/m3 air.Inhalation exposure of rats to Na2H2EDTA 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. 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. Under the current test conditions, the No Observed Adverse Effect Concentration (NOAEC) for local effect in larynx was 3 mg/m3, the NOEC for systemic effects is >15 mg/m³. The local key effect of respirable Na2H2EDTA in the respiratory tract (larynx) is assumed to be mainly concentration-related, hence the impact of exposure time should be low at subcritical concentrations, which was confirmed by the 90d study: although the number of exposures was a factor 13 higher than in the 5d dose-range-finder study, the local effects at 15 mg/m3 in the subchronic inhalation study were comparably mild compared to the 5d dose-range-finder study that showed a more severe effect at 30 mg/m3. It is justified to classify Na2H2EDTA and thus DTPA for repeated dose effects due to the low concentrations of Na2H2EDTA that induce the local effects and a potential human relevance of the effects. However, due to the low severity and incidence of the effects at 15 mg/m3 in the subchronic toxicity study and the reversibility of the local effects, a STOT RE Cat.2 (H373) is considered sufficiently conservative.

Read across is proposed both in choice of the point of departure and the assessment factors applied, for derivation of the DNEL for repeated dose inhalation toxicity endpoints from a recently completed OECD 413 and GLP compliant 90-day inhalation study in rats involving Na2H2-EDTA (CAS no. 139-33-3) to the DTPA category of similar substances (Na5-DTPA (CAS no. 140-01-2), K5-DTPA (CAS no. 7216-95-7) and H5-DTPA (CAS no. 67-43-6)). The principal basis for read across includes the following considerations:

Data from Na2H2-EDTA is considered appropriate for read across to the DTPA category (Na5-DTPA, K5-DTPA and H5-DTPA) since the mode of action for these effects is considered to be related to the chelation of calcium and both chelating agents have a similar affinity for calcium. See the Table below.

Stability constants (Log K values)

Metal ion

EDTA

DTPA

Mg 2+

8.8

9.3

Ca 2+

10.7

10.8

Mn 2+

13.9

15.2

Fe 2+

14.3

16.2

Zn 2+

16.5

18.2

Cu 2+

18.8

21.2

Fe 3+

25.1

28.0

Values are based on Martell AE, Smith RM, NIST Critically selected stability constants of metal complexes (NIST standard reference database 46, Version 7.0, 2003)

The sites of irritation observed in the respiratory tract of rats in the 90-day inhalation toxicity study with Na2H2-EDTA are consistent with the areas where a high degree of test material impaction would occur. Upon impaction, EDTA complexes with calcium and perhaps zinc, removing it from cell junctions and membranes. The removal of these compounds from intercellular junctions causes cells to detach from one another resulting in cell shedding. This leads to tissue regeneration and metaplasia in the affected areas. As cells which have become detached die, inflammation occurs leading to signs of necrosis and infiltration of inflammatory cells. This effect is similar to that observed in the intestines of rats given high oral bolus doses of chelating agents such as EDTA and DTPA.

Given the effects of the substances are similar in nature, involve the same mode of action, are concentration dependent, threshold mediated and do not involve metabolic processes, it is therefore considered relevant to read across from Na2H2-EDTA to the DTPA category of substances (Na5-DTPA, K5-DTPA and H5-DTPA) for the inhalation endpoint.

Differences in molecular weight within the EDTA-category would normally justify correction of long-term inhalation DNELs (local, workplace) on the basis of molecular weight. However the higher molecular number of EDTA versus DTPA at similar concentrations is countered by the generally higher binding affinity and results in similar overall chelating activities of the substances at physiological pH.In addition to this, the effects observed at the highest concentration tested in the study were limited to slight histopathological changes in the epiglottis and larynx in 1 or 2 female animals despite concentration levels tested being 5-fold greater than the point of departure for deriving the inhalation DNEL.Furthermore, the vapour pressure of DTPA is very low, and as such these substances would be considered of low category of concern under REACH with likely negligible exposures.

Taken together the proposed approach to deriving the DNEL from the 90-day inhalation study involving Na2H2-EDTA is considered sufficiently conservative for the DTPA category of substances (Na5-DTPA, K5-DTPA and H5-DTPA) and given the slight nature of the effects observed and the low potential for exposure the need for additional assessment factors for read across is not warranted.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Most relevant endpoint for selection, study is GLP and OECD 413 guideline compliant

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Most relevant endpoint for selection, study is GLP and OECD 413 guideline compliant

Repeated dose toxicity: inhalation - systemic effects (target organ) respiratory: larynx; respiratory: lung

Justification for classification or non-classification

Based on the results obtained in the inhalation toxicity studies and taking into account the provisions laid down in Council Directive 67/548/EEC and CLP, a classification as Xn (R48/20) and STOT RE Cat 2 (H373) is considered justified