Trisodium hydrogen ethylenediaminetetraacetate

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2009-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity: 28/14-Day)

Deviations:

yes

Remarks:

Dosing until Day 5 only

GLP compliance:

yes (incl. QA statement)

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 7 weeks (approx)
Identification: Tattooing of ears
All animals free of disease and clinical signs
Rats housed together (5 animals per cage) in Polysulfon cages
Bedding: Type Lignocel fibres, dust free bedding
Woodne gnawing blocks for enrichment
Rooms: Fully ariconditioned, temperature range 20 to 24 degrees celcius, 30 to 70% humidity
Light/dark cycle of 12 hours (6 am to 6pm light, 6pm to 6 am dark)
Food, drinking water and bedding/enrichment materials were analysed for chemical and microbiological contaminants.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2 - <= 2.7 µm

Remarks on MMAD:

Geometric standard deviation (GSD): 2.0 - 2.22

Details on inhalation exposure:

A dust aerosol was generated using a dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed into the inhalation system. The test substance was mixed with Aerosil R972 prior to facilitate aerosol generation.

The inhalation atmosphere was maintained inside aerodynamic exposure systems consisting of cylindrical inhalation chamber made of stainless steel sheeting and cone shaped outlets and inlets. The rats were restrained in glass exposure tubes with their snouts projecting into the inhalation chamber.
The animals did not ave access to feed or water during the exposure period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of the inhalation atmospheres were analyzed using gravimetry. Daily means were calculated based on 2 measured samples per concentration and exposure. From the Daily mean values of each concentration, mean concentrations and standard deviations were derived.
Consistency of concentrations in each inhalation system was continuously monitored using scattered light photometry.
Particle size analysis was conducted using a cascade impactor.

Duration of treatment / exposure:

Exposures: 6 hours per day
High dose group (1000 mg/m³) was exposued for 1 day only
All other groups were exposed for 5 consecutive days

Frequency of treatment:

daily

Dose / conc.:

30 mg/m³ air (nominal)

Remarks:

actual: 33.3 (± 2.3) mg/m³

Dose / conc.:

300 mg/m³ air (nominal)

Remarks:

actual: 320 (± 27) mg/m³

Dose / conc.:

1 000 mg/m³ air (nominal)

Remarks:

actual: 1103 (± 52) mg/m³

No. of animals per sex per dose:

10 animals per dose group
An additional 10 animals for the high dose group and control

Control animals:

yes, sham-exposed

Details on study design:

The animals were exposed to a respirable dust aerosol for 6 hours per day for 5 consecutive days. The exception was the high dose group (1000 mg/m³) where exposure was for one day only due to mortality observed.
In the control, low and mid dose groups, 5 animals were sacrificed on the day after the last exposure period and 5 were sacrificed 17 days after the last exposure.
10 additional control animals and the 14 surviving high dose group animals were sacrificed on day 14 of the study (14 days after first exposure).

Observations and examinations performed and frequency:

Mortality:
- observations of toxicity or mortality were made twice daily (week days) and once daily (weekends and public holidays)

Clinical Observations:
- Once during the pre-flow period and on post-exposure days. At least three times (before, during and after exposure) on exposure days.

Bodyweight:
- Start of pre-flowm start of exposuer period, once per week prior to necropsy

Food consumption:
- Determined weekly and calculated as mean food consumption in grams per animal per day (no statistical evaluation possible due to grouphousing conditions)

Clinical Pathology:
- On day of sacrifice, blood aken from retro-orbital venous plexus fromfasted animals.

Hematology:
- Leucocyte count, erthyrocyte count, hemoglobin, hematocrit, MCV, MCH, MCHC, Platelet count, differential blood count, reticulocytes count, clotting anlyses (prothrombin time)

Clinical Chemistry:
- ALT, AST, ALP, GGT

Blood chemistry:
- sodium, potassium, chloride, inorg. phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, magnesium

Sacrifice and pathology:

5 animals per dose group were sacrificed and underwent gross macroscopic examination the day after the last exposure.
5 animals from each dose group (control, 30, and 300 mg/m³) were sacrificed at the end of the recovery period.
All animals dying prematurely were examined histologically.
Surviving animals in the 1000 mg/m³ group and 10 control animals were sacrificed and examined on day 14.

Necropsy:
- all animals were necropsied and assessed for gross pathology. Animals dying intercurrently or were killed in a moribund state wee necropsied as soon as possible after their death.

Organ Weights:
- Adrenals, brain, epididymides, heart, liver, kidneys, lungs, spleem, testes, thymus thyroid glands

Organ/tissue fixation:
- All gross lesions, adrenal glands, brain with olfactory bulb, bone marrow (femur), eyes with optic nerve, heart, kidneys, liver, larynx/pharynx, lymph nodes, nose, oesophagus, seminal vesicle, spinal cord, stomach (forestomach and glandular), spleen, testes, thyroid, thymus, trachea, urinary bladder
- One lobe of the liver from the lobus Dexter medialis and the lobus sinster lateralis was fixed in Carnoy's solution and embedded in paraplast. All other tissues were fixed in formulin.

Histology (All animals per group):
- All gross lesions, nasal cavity (4 levels), larynx (3 levels), trachea, lungs (5 lobes), lymph nodes (tracheobronchial and mediastinal), kidneys and liver

Statistics:

Body weight/body weight change, food consumption - comparison of each group with control using DUNNETTS test (two-sided) for the hypothesis of equal means
Clinical pathology, urine volumes, urine specific gravity, Weight parameters - Non-parametric one-way analysis using Kruskal-wallis test (two sided). If resulting p-value was less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two sided) for equal means.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Mortality:

mortality observed, treatment-related

Description (incidence):

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased bodyweight change in mid and high dose group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

decreased food consumption between days 0 and 1 in mid and high dose group

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Lung weight increased in low and mid dose group

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Congestion, edema and multifocal hemorraghes in lungs of high dose group

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See details on results below.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Histopathology results:
High dose: Multifocal hemorraghes in the lungs; Inflammatory cell infiltrates

Mid dose:
Larynx: laryngeal, epithelial necrosis, multifocal, in various levels of the larynx
Inflammatory cell infiltrates in various levels of the larynx
laryngeal squamous metaplasia, multifocal, in various levels of the larynx
Regenerative hyperplasia of the laryngeal epithelium, multifocal, in various levels of the larynx
Lungs: Regenerative hyperplasia of bronchiolar epithelium (predominantly: medium bronchi, terminal bronchioles)
Mucous cell hyperplasia in large bronchi
interstitial infiltration of eosinophylic granulocytic cells

Low dose:
Larynx: Laryngeal, epithelial necrosis, multifocal, at the base of the epiglittis (level 1)
Inflammatory cell infiltrates at the base of the epiglottis (level 1)
Lungs: Regenerative hyperplasia of the bronchiolar epithelium (predominantly medium bronchi and terminal bronchioles)
Mucous cell hyperplasia in large bronchi
interstitial infiltration of eosinophylic granulocytic cells.

There were no histopathological findings in any of the recovery group animals. Thus all pathology was reversible within the recovery period.

Key result

Dose descriptor:

LOAEC

Remarks:

local effects

Effect level:

30 mg/m³ air (nominal)

Based on:

act. ingr.

Remarks:

Na2H2EDTA

Sex:

male

Basis for effect level:

other: histopathology of the respiratory tract and lung weights

Key result

Dose descriptor:

NOAEC

Remarks:

systemic effects

Effect level:

30 mg/m³ air (nominal)

Based on:

act. ingr.

Remarks:

Na2H2EDTA

Sex:

male

Basis for effect level:

other: clinical signs, reduced food consumption and body weights at 300 mg/m3

Critical effects observed:

not specified

Conclusions:

In this 5-day inhalation study in rats, a NOAEC for local effects could not be established due to histopathological findings in the lungs noted at the lowest exposure concentration. A single inhalation exposure to 1000 mg/m³ disodium hydrogen EDTA for 6 hours caused lethality in 6 out of 20 male rats. Histological examination of the lung of the dead rats revealed congestion, edema, multifocal hemorraghes and inflammatory cell infiltrates. Inhalation exposure of rats to disodium dihydrogen EDTA for 6 hours per day on 5 consecutive days caused 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 for local effects could not be determined and the LOAEC for local effects was considered to be 30 mg/m3. A NOAEC for systemic toxicity was established at 30 mg/m3.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 412 (Repeated Dose Inhalation Toxicity: 28/14-Day)

Deviations:

yes

Remarks:

Dosing until day 5 only

GLP compliance:

yes (incl. QA statement)

Test type:

other: subacute

Test material

Test material information

Constituent 1

Reference substance name:

Disodium dihydrogen ethylenediaminetetraacetate

EC Number:

205-358-3

EC Name:

Disodium dihydrogen ethylenediaminetetraacetate

Cas Number:

139-33-3

Molecular formula:

C10H16N2O8.2Na

IUPAC Name:

disodium dihydrogen 2,2',2'',2'''-(ethane-1,2-diyldinitrilo)tetraacetate

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No. of test material: 06088797V0

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Expiry date: 1 September 2011

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

Age: 7 weeks (approx.)
Identification: Tattooing of ears
All animals free of disease and clinical signs
Rats housed together (5 animals per cage) in Polysulfon cages
Bedding: Type Lignocel fibres, dust free bedding
Wooden gnawing blocks for enrichment
Rooms: Fully airconditioned, temperature range 20 to 24 °C, 30 to 70 % humidity
Light/dark cycle of 12 hours (6 am to 6 pm light, 6 pm to 6 am dark)
Food, drinking water and bedding/enrichment materials were analysed for chemical and microbiological contaminants.

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2 - <= 2.7 µm

Geometric standard deviation (GSD):

>= 2 - <= 2.22

Details on inhalation exposure:

A dust aerosol was generated using a dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed into the inhalation system. The test substance was mixed with Aerosil R972 prior to facilitate aerosol generation.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Concentrations of the inhalation atmospheres were analyzed using gravimetry. Daily means were calculated based on 2 measured samples per concentration and exposure. From the Daily mean values of each concentration, mean concentrations and standard deviati

Duration of exposure:

6 h

Remarks on duration:

daily, 5 consecutive days

Concentrations:

- 30, 300, 1000 mg/m³ (nominal conc. of Na2H2EDTA)
- 33.3 (± 2.3), 320 (± 27), 1103 (± 52) mg/m³ (measured (with SD) referring to test substance Na2H2EDTA × 2 H2O)

No. of animals per sex per dose:

10 animals per dose group
An additional 10 animals for the high dose group and control

Control animals:

yes

Details on study design:

The animals were exposed to a respirable dust aerosol for 6 hours per day for 5 consecutive days. The exception was the high dose group (1000 mg/m³) where exposure was for one day only due to mortality observed.

Statistics:

Body weight/body weight change, food consumption - comparison of each group with control using DUNNETTS test (two-sided) for the hypothesis of equal means
Clinical pathology, urine volumes, urine specific gravity, Weight parameters - Non-parametric one-way analysis using Kruskal-wallis test (two sided). If resulting p-value was less than 0.05, a pairwise comparison of each dose group with the control group was performed using Wilcoxon-test (two sided) for equal means.

Results and discussion

Effect levels

Key result

Sex:

male

Dose descriptor:

other: LOAEC

Effect level:

ca. 30 mg/m³ air

Based on:

act. ingr.

Remarks:

Na2H2EDTA

Remarks on result:

other: Basis for effect level: histopathology of the respiratory tract and lung weights

Mortality:

6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Clinical signs:

other: 6 deaths in the high dose group on days 0 and 1. Accelerated respiration, respiration sounds, piloerection, red encrusted nose, hunched position; Mid dose group - accelerated respiration, respiration sounds, piloerection, reduced fur care

Body weight:

Decreased bodyweight change in mid and high dose group

Gross pathology:

Congestion, edema and multifocal hemorraghes in lungs of high dose group

Other findings:

FOOD CONSUMPTION
- decreased food consumption between days 0 and 1 in mid and high dose group

ORGAN WEIGHTS
- Lung weight increase in low and mid dose group

Any other information on results incl. tables

DETAILS ON RESULTS

Histopathology results:

High dose: Multifocal hemorraghes in the lungs; Inflammatory cell infiltrates

Mid dose:

Larynx: laryngeal, epithelial necrosis, multifocal, in various levels of the larynx

Inflammatory cell infiltrates in various levels of the larynx

laryngeal squamous metaplasia, multifocal, in various levels of the larynx

Regenerative hyperplasia of the laryngeal epithelium, multifocal, in various levels of the larynx

Lungs: Regenerative hyperplasia of bronchiolar epithelium (predominantly: medium bronchi, terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells

Low dose:

Larynx: Laryngeal, epithelial necrosis, multifocal, at the base of the epiglittis (level 1)

Inflammatory cell infiltrates at the base of the epiglottis (level 1)

Lungs: Regenerative hyperplasia of the bronchiolar epithelium (predominantly medium bronchi and terminal bronchioles)

Mucous cell hyperplasia in large bronchi

interstitial infiltration of eosinophylic granulocytic cells.

There were no histopathological findings in any of the recovery group animals. Thus all pathology was reversible within the recovery period.

Applicant's summary and conclusion

Interpretation of results:

other: CLP/GHS criteria met; classification required as Acute Tox 4, H332 according to Regulation (EC) No. 1272/2008

Conclusions:

In this acute inhalation toxicity study with the source substance disodium hydrogen EDTA (CAS 139-33-3) the acute inhalation LOAC was found to be 30 mg/m3.

CLP: Acute Tox 4, H332