Diammonium dihydrogen ethylenediaminetetraacetate

Endpoint summary

Description of key information

No studies on the acute toxicity of diammonium dihydrogen ethylenediaminetetraacetate are available. Like the sodium salts of EDTA, ammonium salt of EDTA are likely to dissociate at physiological pH to the EDTA anion and the ammonium ion, therefore the acute oral toxicity data of EDTA free acid and its sodium salts have be used for an estimation of acute toxicity of diammonium dihydrogen EDTA. The acute oral LD50 values were 2800 mg/kg bw (Na2EDTA) and 4500 mg/kg bw (EDTA free acid), respectively. The LOAEC established in an inhalation study with Na2EDTAwas considered being 30 mg/m³ air. Furthermore it is highly unlikely that EDTA induced acute dermal toxicity as neither Ca or Na salts of EDTA are able to penetrate the skin.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

Body weight was only determined at the beginning of the study (OECD: weekly); Observation period: 7 days (OECD:14 days)

Principles of method if other than guideline:

BASF-TEST: In principle, the methods described in the OECD Guideline 401 were used. Young adult laboratory rats were purchased from breeder. Usually the source and strain of animals were not documented. Several groups of 5 rats per sex and dose were treated simultaneously by gavage with preparations of the test substance in suitable vehicle. The concentrations of these preparations were used to achieve comparable volumes per kg body weight. Group-wise documentation of clinical signs was performed over the 7 day study period. Body weight was determined before the start of the study only, as it was needed for determination of dose.

GLP compliance:

no

Test type:

standard acute method

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Mean body weight at study initiation:
259 g males/ 211 g females

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

DOSAGE PREPARATION:
- Stock solutions prepared: 30%
- Dose volume applied:
2500 mg/kg bw dose group: 8.33 mL/kg bw
3200 mg/kg bw dose group: 10.66 mL/kg bw
4000 mg/kg bw dose group: 13.33 mL/kg bw
5000 mg/kg bw dose group: 16.66 mL/kg bw
6400 mg/kg bw dose group: 21.4 mLkg bw

Doses:

2500; 3200; 4000; 5000; 6400 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 7 days;
- The animals were observed for mortality and clinical signs of toxicity;
- Frequency of observations: Several times on the application day, thereafter once each working day;
- Body weights were only recorded at the beginning of the study;
- Necropsy of survivors and animals which died performed: yes

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 800 mg/kg bw

Mortality:

- One female died in the 2500 mg/kg bw dose group; 9/10 animals died in the 3200 mg/kg bw dose group and all animals of the higher dose groups (see table 1).

Clinical signs:

other: - 2500, 3200 and 4000 mg/kg bw: directly after application: accelerated respiration, squatting posture, twitching, ataxia, red eyes, some animals showed light secretion, reluctance to move; the next day: squatting posture, contaminated fur, intermittened

Gross pathology:

Animals which died:
- heart: acute dilatation, venous hyperemia
- liver: congestion
- gut: diarrhea like content
- stomach: dilatation
- kidneys: degeneration
Animals which were sacrificed:
- nothing abnormal detected

Table 1: Mortalities of rats after oral application

		2500 mg/kg bw	3200 mg/kg bw	4000 mg/kg bw	5000 mg/kg bw	6400 mg/kg bw
1 h	male	0/5	0/5	0/5	0/5	0/5
	female	0/5	0/5	0/5	0/5	0/5
24 h	male	0/5	3/5	5/5	5/5	5/5
	female	1/5	5/5	5/5	5/5	5/5
48 h	male	0/5	3/5	5/5	5/5	5/5
	female	1/5	5/5	5/5	5/5	5/5
7 d	male	0/5	4/5	5/5	5/5	5/5
	female	1/5	5/5	5/5	5/5	5/5

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 800 mg/kg bw

Quality of whole database:

similar to OECD TG 401 (Read Across)

Acute toxicity: via inhalation route

Link to relevant study records

Reference

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

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)

Test type:

other: subacute

Limit test:

yes

Specific details on test material used for the study:

Batch Number: 06088797V0
Expiry date: 01 September 2011

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 7 weeks (approx.)
- Housing: housed together (5 animals per cage) in Polysulfon cages:Type Lignocel fibres, dust free
bedding; Wooden gnawing blocks for enrichment
ENVIRONMENTAL CONDITIONS
Rooms: fully air-conditioned
- Temperature (°C): 20 to 24°C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): cycle of 12 hours (6 am to 6pm light, 6pm to 6 am dark)
Identification: Tattooing of ears
All animals free of disease and clinical signs. 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

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 SD

Duration of exposure:

6 h

Remarks on duration:

per day, 5 consecutive days

Concentrations:

- 30, 300, 1000 mg/m³ (nominal conc.)

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.

Key result

Sex:

male

Dose descriptor:

other: LOAEC

Effect level:

ca. 30 mg/m³ air

Based on:

test mat.

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

HISTOPATHOLOGY
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.

Interpretation of results:

Category 4 based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Value:

30 mg/m³ air

Quality of whole database:

OECD TG 412 (Read Across)

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

oral route:

In a study conducted by BASF AG (1973) single doses of 3200, 4000, 5000, 6400, 8000 and 10000 mg/kg bw edetic acid were administered by gavage to male and females rats as 30% solution in a carboxymethyl cellulose solution. The dose groups consisted of 5 males and 5 females each and the animals were observed for 14 days. The LD50 was found to be 4500 mg/kg bw. Clinical symptoms were: squatting posture, aggressiveness, diarrhea and contaminated fur in all dose group. Autopsy of the animals which died revealed acute heart dilatation, bloody ulceration of the stomach and soft to fluid contents of the intestine. Additionally single doses of 2500, 3200, 4000, 5000 and 6400 mg/kg bw Na2EDTA were administered by gavage to male and female rats as 30% solution in carboxymethyl cellulose solution (BASF, 1973). The dose groups consisted of 5 males and 5 females each and the animals were observed for 7 days. The LD 50 was found to be 2800 mg/kg bw. Clinical symptoms were accelerated respiration, squatting posture, contaminated fur, intermittened respiration, reluctance to move. Autopsy of animals which died revealed dilatation of the heart and stomach as well as diarrhea like content in the gut, congestion of the liver and degeneration of the kidney. The animals which were sacrificed showed no abnormalities during necropsy.

Inhalation route:

In a subacute repeated dose toxicity study (BASF, 2009) 10 male Wistar rats per dose were exposed to a respirable dust aerosol of Na2H2EDTA (CAS 139-33-3) for 6 hours per day for 5 consecutive days at concentrations of 0, 30, 300, 1000 mg/m³ air (also see chapter 7.5). Exposure in the high dose group (1000 mg/m3) was for one day only due to mortality observed. Inhalation exposure to 1000 mg/m³ disodium 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 EDTA for 6 hours per day, 5 consecutive days cause concentration dependent lesions in the larynx and lungs that were fully reversible within 14 days. Due to histopathological changes in the low dose group a no observed effect level could not be determined. The LOAEC was considered to be 30 mg/m³ air.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the results of a repeated dose toxicity study (5 consecutive days, see chap. 7.5.3) a classification for acute inhalation toxicity (Cat. 4; H332:"Harmful if inhaled") is done under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179. A classification is not warranted in respect to acute oral and dermal toxicity.