N-carboxymethyliminobis(ethylenenitrilo)tetra(acetic acid)

Endpoint summary

Description of key information

One 28-day oral gavage study in rats using pentapotassium DTPA, one 28-day oral, drinking water study using pentasodium DTPA, one 90-day and one 5-day inhalation study using disodium EDTA. Additional information comes from published literature on other salts of DTPA such as zinc and calcium.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes (incl. QA statement)

Remarks:

testing lab.

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Male and female Wistar rats (Chbb:THOM (SPF)) supplied by Dr . Karl Thomae GmbH, Biberach/Riss, Germany, which were free of signs of disease were used for the investigations.
The rats were identified clearly by tattooing of the respective animal number into the ears.
The rats were housed singly in type DK III stainless steel wire mesh cages supplied by Becker & Co., Castrop-Rauxel, Germany (floor area about 800 cm2). Underneath the cages, waste trays were fixed containing absorbent material (type 3/ dustfree embedding, supplied by SSNIFF, Soest, Germany). The animals were housed in a fully air-conditioned room. Central air-conditioning guaranteed a range of 20 - 24°C for temperature and of 30 - 70% for relative humidity. The day/night rhythm was 12 hours (12 hours light from 06.00 a.m. - 06.00 p.m., 12 hours dark from 06.00 p.m. - 06.00 a.m.). Deviations from these ranges did not occur. The animal room was completely disinfected using a disinfector ("AUTEX", fully automatic, formalin-ammoniabased
terminal disinfector). The floor and the walls were cleaned once a week. The cleansing liquid used was water containing about 0.1 % Incidin perfect® (supplied by Henkel, Düsseldorf, Germany). The food used was basic maintenance diet rat/mouse/hamster, meal, supplied by Klingentalmühle AG, Kaiseraugst, Switzerland. Food and drinking water (from water bottles) were available ad libitum.

Route of administration:

oral: drinking water

Details on oral exposure:

The test substance was administered as a solution in drinking water.
The test substance was weighed for the specific test group, and the appropriate amount of drinking water was added (also weighed). This mixture was subsequently stirred with a magnetic stirrer for at least 30 minutes to reach complete solubility of the test substance in the drinking water. The drinking water solutions were prepared twice a week (Monday to Friday).
Before the beginning of the study, the stability of the test substance in the vehicle over a period of 4 days at room temperature was ordered. At the start of the study one sample of each concentration was sent to the analytical laboratory for determination of the correctness of the concentration of the test substance preparations.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability of the test substance in the vehicle over a period of 4 days at room temperature and the correctness of the concentrations were verified analytically.

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
600 ppm; 3,000 ppm and 12,000 ppm
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

Selction of the doses for this study was based on the results of a previous palatability study.
In this study, Trilon C flüssig was administered in the drinking water to 3 male and 3 female Wistar rats per group for 2 weeks .
Following substance-induced changes were observed:
15,000 ppm:
• Discoloration of the feces in both sexes
• Decreased food consumption in both sexes
• Decreased water consumption in both sexes
• Piloerection and reduced general state in 1 male and 1 female
• Diminished adipose tissue in 1 male and 2 females
5,000 ppm:
• Discoloration of the feces in both sexes
Therefore, the following doses were chosen for the 4-week administration in the drinking water (doses of active ingredient):
600 ppm: as the lowest dose level
3,000 ppm: as the intermediate dose level
12,000 ppm: as the high dose, resulting in a substance intake of > 1,000 mg/kg body weight

Positive control:

none

Observations and examinations performed and frequency:

Clinical observations: A check was made twice Mondays to Fridays and once a day on Saturdays, Sundays and public holidays for general observations. Once a week an additional comprehensive clinical examination was carried out. In order to prepare tables of the clinical symptoms observed, the data were entered offline into the computer systems; according to the particular health status of the individual animals, sometimes several, different clinical signs were documented during one week.
Mortality: A check was made twice Mondays to Fridays and once a day on Saturdays, Sundays and public holidays for any dead or moribund animals.
CLINICAL PATHOLOGY: Blood was taken from the retroorbital venous plexus in the morning from fasted animals without anesthesia. The blood sampling procedure and the subsequent analysis of the blood and serum samples were carried out in a randomized sequence.
The following parameters were determined in blood with EDTA-K3 as anticoagulant using a particle counter (S Plus model, by Coulter, Krefeld, Germany) :
• leukocytes
• erythrocytes
• hemoglobin
• hematocrit
• mean corpuscular volume
• mean corpuscular hemoglobi n
• mean corpuscular hemoglobin concentration
• platelets
Clinical chemistry: The following parameters were determined :
• alanine aminotransferase
• aspartate aminotransferase
• alkaline phosphatas e
• serum-y-glutamyltransferase
• sodium
• potassium
• chlorid e
• inorganic phosphate
• calcium
• urea
• creatinine
• glucose
• total bilirubin
• total protein
• albumin
• globulins
• triglycerides
• cholesterol
• magnesium
Urinalyses: The following examinations were carried out:
• volume
• color
• turbidity
• nitrite
• pH
• protein
• glucose
• ketones
• urobilinogen
• bilirubi n
• blood
• specific gravity
• sediment

Sacrifice and pathology:

A check was made twice Mondays to Fridays and once a day on Saturdays, Sundays and public holidays for any dead or moribund animals.
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Statistics for clinical examinations, clinical chemistry and hematology, urinalyses.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

no effects observed

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

12,000 ppm (about 1,775 mg/kg body weight): discoloration of the feces in both sexes; decreased food consumption in both sexes; significantly decreased body weight, resulting in reduced values of about 22% (males) and 7% (females) at the end of the study; significantly reduced body weight change, resulting in reduced values of about 46% (males) and 21 % (females) at the end of the study; increase in alanine aminotransferase in the males; increase in specific gravity, renal epithelial cells and casts in the urines of individual males; dark yellow colored urines in individual males; decrease in urine volume in both sexes; decrease in alkaline phosphatase in the females; transitional cell hyperplasia in the urinary bladder of 4 male and 2 female test animals.
3,000 ppm (about 420 mg/kg body weight): significantly decreased body weight change in the males in the last test week (about 10% below controls); increase in alanine aminotransferase in the males; decrease in alkaline phosphatase in the females.
600 ppm (about 75 mg/kg body weight): no substance-induced changes were detected.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 75 mg/kg bw (total dose)

Sex:

male/female

Basis for effect level:

other: Clear changes in body weight, clinical chemistry, urinalaysis, and histopathology in the high dose group. Clear changes in body weight and clinical chemistry in the mid dose group.

Critical effects observed:

not specified

Conclusions:

Clear effects of body weight and histopathological changes of the urinary tract with corroborating results of the urinalyses were obtained at 12,000 ppm. At 3,000 ppm, only minor effects were obtained. Although the clinicochemical findings at 12,000 and 3,000 ppm are difficult to explain, they must be assessed as substance-related. A clear "no observed adverse effect level" was obtained at 600 ppm (about 75 mg/kg body weight) under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

See read across argumentation in section 7 (DNEL setting).

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

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)

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, 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:

NOAEC

Effect level:

3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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.

Conclusions:

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

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 the larynx is 3 mg/m3, the NOEC for systemic effects is 15 mg/m³.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

15 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

See read across argumentation in section 7 (DNEL setting) and read across document in section 13.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

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)

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, 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:

NOAEC

Effect level:

3 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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.

Conclusions:

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

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 the larynx is 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

Quality of whole database:

See read across argumentation in section 7 (DNEL setting) and read across document in section 13.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Oral

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 via the oral route.

Inhalation

To date, two guideline studies have been performed to evaluate the potential toxicity of chelating agents following repeated inhalation exposure. A 5 -day inhalation study with a structurally related compound EDTA-Na2H2 showed histopathological changes in the respiratory tract at a concentration of 30 mg/m3. After a recovery period of 14 days, all changes had disappeared.The 90-day inhalation study with the same substance was carried out according to OECD 413 and EC No 440/2008. Wistar rats, 10 per sex per test group, were exposed (head-nose only), to dust aerosol for 6 hours per day, on 5 consecutive days per week for 13 weeks (65 exposures). The target concentrations were 0.5, 3 and 15 mg/m3. A concurrent control group was exposed to air. On each exposure day a clinical examination was performed before, during and after exposure. Detailed clinical observation was performed at the beginning, midterm and end of the study. Ophthalmology was performed before the beginning of the exposure in all test groups and at the end of the end of the exposure in the control and high concentration group animals. Body weights and food consumption of the animals were determined weekly. At the end of the exposure period, functional observation battery and motor activity tests were performed. On the day after the last exposure, blood was sampled and examined for a range of hematology and clinical chemical parameters as indicated in the guideline. After blood sampling the animals were sacrificed and subject to necropsy (including macroscopic examination of the major internal organs and collection of organ weight data). Selected tissues were processed histopathologically and were evaluated by light microscopy according to the OECD guideline. When compared with the control group, the following treatment-related adverse findings were noted in Wistar rats after 90 days of inhalation:

High concentration (15 mg/m³)

· Focal hyperplasia of the laryngeal epithelium at the base of the epiglottis in one female animal

· Slight granulocytic infiltrates at the base of the epiglottis of the larynx in two female animals

Mid (3 mg/m³) and low concentration (0.5 mg/m³)

No adverse findings.

Conclusion

Inhalation exposure of rats to EDTA-Na2H2 for 90 days (65 exposures) did not lead to any substance related clinical signs of toxicity. Nor were there any effects in clinical chemistry and 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 effects was 3 mg/m3, the NO(A)EC for systemic effects was 15 mg/m³. Inhalation exposure of rats to EDTA-Na2H2 for 6 hours per day, 5 consecutive days causes concentration dependent lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopahological changes in the low-dose group a no observed effect level could not be determined.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

Study available for DTPA-Na5; supported by study with DTPA-K5 (NOAEL of 83 mg/kg bw).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:

Well performed 90-day inhalation study with structurally-related substance.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:

Well performed 90-day inhalation study with structurally-related substance.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver; urogenital: kidneys

Justification for classification or non-classification

Classification for repeated dose toxicity is proposed as the CLP criteria for classification for target organ toxicity have been met. According to the guidance on the application of CLP criteria (Version 4.0, 2013),substances that, on the basis of evidence from studies in experimental animals can be presumed to have the potential to be harmful to human health following repeated exposures should be classified as STOT RE. For DTPA-H5, given the available evidence from a structuraly related substance, with only slight histopathological effects at the larynx at a concentration of 15 mg/m3, it would be appropriate to apply Category 2, H373, specifically for the inhalation route.