Disodium [[N,N'-ethylenebis[N-(carboxymethyl)glycinato]](4-)-N,N',O,O',ON,ON']cuprate(2-)

Endpoint summary

Description of key information

The oral LD50 value in rats was 890 mg/kg bw; the 4-h LC50 value is in excess of 5.30 g/m3.

No acute dermal toxicity studies are available but based on read across (see also section 13), acute dermal toxicity following exposure to EDTA-CuNa2 is not expected.  

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

Study period:

January-February 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Although no guideline is mentioned, the study was carried out according to the old version of the OECD guideline 403 (1981).

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Doses:

464, 681, 1000, 1470, 2150 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Sex:

male/female

Dose descriptor:

LD50

Effect level:

890 mg/kg bw

Based on:

test mat.

95% CL:

760 - 1 040

Sex:

female

Dose descriptor:

LD50

Effect level:

830 mg/kg bw

Based on:

test mat.

95% CL:

610 - 1 120

Sex:

male

Dose descriptor:

LD50

Effect level:

ca. 1 000 mg/kg bw

Based on:

test mat.

Mortality:

Yes, see below

Clinical signs:

other: Yes, see below

Gross pathology:

Yes, see below

Mortality males:

464 mg/kg: 0%

681 mg/kg: 0%

1000 mg/kg: 60%

1470 mg/kg: 100%

2150 mg/kg: 100%

Mortality females:

464 mg/kg: 0%

681 mg/kg: 20%

1000 mg/kg: 80%

1470 mg/kg: 100%

2150 mg/kg: 100%

Observations in animals that died:

- 681 mg/kg: general congestive hyperaemia, strikingly dark, black-brown kidneys, light bronze-coloured liver, green/black contents in caecum

- 1000 mg/kg: haematin-containing contents in intestines, liver with grey lobular periphery, in some cases confluent

- 1000 -2150 mg/kg: in stomach: suspicion of irritation, blood ulceration, corrosion of the glandubular stomach mucosa, mucosa stained in several cases by test substance; in small intestines contents stained with substance; strikingly liquid coontents in caecum

- 2150 mg/kg: substance-stained urine

Histopathology:

- 681 mg/kg (one animal): panlobular fatty degeneration in liver and renal tubular necroses in cortex and hydropic tubular degeneration in the medulla

- 1000 mg/kg (one animal): acute yellow dystrophy in the liver

Observations in sacrificed animals:

- 464 and 1000 mg/kg: no abnormalities

- 681 mg/kg: enlarged kidneys in 2 males, pale sreen-type pattern, in other animals no abnormalities

Histopathology:

- 681 mg/kg (one animal): nephrocalcinosis in the cortex and medulla of the kidneys, interstitial repair processes, tubular epithelial regeneration

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Based on the results of this study, the acute oral LD50 value was 890 mg/kg bw in male and female rats combined.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

890 mg/kg bw

Quality of whole database:

Three studies available.

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

Study period:

August 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Deviations:

yes

Remarks:

particle size slightly outside the required range

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

On the exposure day, the rats were about 8 weeks old.
Body weight variation did not exceed ± 20% of the mean weight for each sex. Mean body weight just before exposure on day 0 was 284 and 233 g for males and females, respectively.
Duration of the acclimatization period: 7 days.
Housing: under conventional conditions in one room separated by sex.
The temperature and relative humidity in the animal room: 22 ± 2°C and at 45-65%, respectively. Occasionally, the relative humidity briefly
(about two hours or less) exceeded 65% after cleaning activities or filling up drinking bottles in the animal room (highest value recorded: 83.3%). In addition, the relative humidity was higher than 65% during three longer periods due to unknown causes (these periods lasted about 4, 10 or 16 hours and occurred on 18-20 August 2012; the highest value recorded was 85.6%).
Lighting: artificial (fluorescent tubes) with a sequence of 12 hours light and 12 hours dark.
Number of air changes: about 10 per hour.

Caging: except during exposure, the animals were housed in groups of three, separated by sex, in macrolon cages (type 4S) with a bedding of wood shavings (Lignocel, Type ¾ from Rettenmaier, Rosenberg, Germany) and a wooden block (from ABEDD, Austria) and strips of paper (Enviro-dri obtained through Lillico, Betchworth, England) as environmental enrichment. During exposure, the animals were housed individually in the exposure unit. Immediately after exposure, the animals were returned to their home cages.

Feed and drinking water were provided ad libitum from the arrival of the animals until the end of the study, except during exposure when they had no access to feed or water. The animals received a cereal-based (closed formula) rodent diet.

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose/head only

Vehicle:

air

Details on inhalation exposure:

To generate the test atmosphere, the test material was first dissolved in demineralized water (42%, w/w) and then nebulized. The solution was prepared, within an hour prior to initiation of test atmosphere generation, by adding the test material (351 g) to a pre-weighed amount of water (488 g) in a 1-L measuring cylinder under continuous stirring on a magnetic stirrer (small vortex).
The solution (amount controlled by a peristaltic pump) was nebulized using an airdriven atomizer. The atomizer was supplied with a stream of compressed dry air. The air pressure on the atomizer was regulated by a reducing valve and the airflow was measured with a mass view meter. The test
atmosphere was introduced at the top of the exposure chamber, directed downward and led to the noses of the animals. At the bottom of the unit the test atmosphere was exhausted. The animals were placed in the exposure unit after stabilization of the test atmosphere (T95 was about 7 minutes). The period between the start of the generation of the test atmosphere and the start of exposure of the animals was 66 minutes.

The actual concentration of the test material in the test atmosphere was measured by gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing approximately 5 L test atmosphere at 5 L/min through fibre glass filters (Sartorius, 13400-47). Filters were weighed before sampling, loaded with a sample of test atmosphere, dried for 15 minutes (which was sufficient to obtain a stable filter weight) and weighed again. The loaded filters were dried by means of a stream of compressed dry air controlled by a mass flow controller (0.125 normal litre/min/filter). It was not necessary to correct the mass of dried test material on the filter for water retained in the crystal structure of the test
material (in a preliminary test, a weighed filter was loaded with a known amount of test material dissolved in water, desiccated with dry air and weighed again; the results indicated that no water was retained in the crystal structure). The actual concentration was calculated by dividing the amount of dried test material present on the filter by the volume of the sample taken. A total number of 12 samples were taken during exposure of the animals.

The nominal concentration was determined by dividing the total amount of test material used (by weight) by the total volume of air passed through the exposure unit. The generation efficiency was calculated from the actual and the nominal concentration (efficiency = actual concentration as percentage of nominal concentration).

Particle size distribution measurements were carried out by means of a 10-stage cascade impactor (Sierra instruments, Carmel Valley, California, USA). During exposure of the animals, two measurements were made. During preliminary test atmosphere generation, one measurement was made at the final settings used during exposure. The Mass Median Aerodynamic Diameter (MMAD) and geometric standard deviation (gsd) were calculated.

The chamber airflow and the temperature and relative humidity of the test atmosphere were recorded eight times during exposure (about twice per hour. A mass view meter was used to measure the airflow. The temperature and relative humidity were measured by means of a RH/T device. The oxygen concentration was checked once during exposure. The carbon dioxide concentration was calculated.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5.30 g/m3 +/- 0.97 g/m3 (n=12)

No. of animals per sex per dose:

3 males and 3 females

Control animals:

no

Details on study design:

On the exposure day, the animals were observed for clinical signs just before exposure, four times during exposure (about once per hour, starting 48 minutes after initiation of treatment), and twice after exposure. During the observation period, each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. On working days, all cages were checked again in the afternoon. In weekends only one check per day was carried out. During exposure attention was directed to breathing abnormalities and restlessness. All abnormalities, signs of ill health, and reactions to treatment were recorded.

The body weight of each animal was recorded on days -1, 0 (just before exposure), 1, 3, 7 and 14 (prior to necropsy). The weight changes over adjacent days and over the 14-day observation period were calculated from the body weight data.

At the end of the 14-day observation period, the animals were sacrificed by exsanguination from the abdominal aorta under pentobarbital anaesthesia. At necropsy, abdominal and thoracic organs were examined in situ for gross pathological changes, with particular attention to any changes in the respiratory tract. The animal that died during the observation period was also subjected to post-mortem examination. After the post-mortem examination, the carcasses were discarded.

Statistics:

Not applicable (limit test)

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.3 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

One male rat (number 20) was found dead on day 2 (early in the morning). The other animals survived until scheduled sacrifice at the end of the 14-day observation period.

Clinical signs:

other: During exposure, all animals showed shallow breathing and decreased breathing rate. Shallow breathing was first seen about one hour after initiation of exposure in a few animals and occurred in all animals at the subsequent observation time points (severi

Body weight:

One day after exposure and between days 1-3, all animals had lost weight (in total about 15% compared to their pre-exposure weight on day 0). Between days 3-7, one male and one female lost some more weight whereas the other survivors gained weight. Between days 7-14, all survivors showed catch-up growth.

Gross pathology:

At necropsy, the male animal that was found dead on day 2 showed dirty fur, poorly collapsed, red lungs, and a red thymus.
Macroscopic examination of the survivors showed enlarged kidneys in two males and various pulmonary changes in one male and all females. The affected lungs were pale, had white patches, a few petechiae and, in females only, indentations. In addition, one female showed a large red patch on two lung lobes. These
gross lesions in the kidneys and lungs were considered to be related to treatment

Other findings:

No

Interpretation of results:

GHS criteria not met

Conclusions:

The 4-h LC50 is in excess of 5.30 g/m3.

Executive summary:

To examine the acute inhalation toxicity of EDTA-CuNa2, three male and three female rats were exposed for four hours to a target limit concentration of 5 g/m3 EDTA-CuNa2. Thereafter, the animals were kept for an observation period of 14 days. To detect adverse effects, clinical observations were made during and after exposure, body weight was measured before exposure (day 0) and at days 1, 3, 7 and 14, and macroscopic examination was conducted at the end of the observation period. The animal found dead on day 2 was also examined macroscopically.

The actual concentration of the test material during exposure was 5.30 ± 0.97 g/m3 (mean ± sd; n=12) based on gravimetric analysis of the test atmosphere. The mass median aerodynamic diameter (MMAD) of the aerosol was 5.38 and 4.85 μm (duplicate measurements) and the distribution of particle sizes had a geometric standard deviation (gsd) of 2.44 and 2.43, respectively. This particle size exceeded the upper limit of the range recommended by OECD guideline 436 (MMAD 1-4 μm). During extensive preliminary testing, it appeared that it was not feasible to generate a respirable (MMAD 1-4 μm) solid aerosol of EDTA-CuNa2 at the limit concentration of 5 g/m3.

One male rat was found dead on the second day after exposure. Necropsy did not reveal a specific cause of death (the animal had dirty fur, poorly collapsed, red lungs and a red thymus). The other animals survived until scheduled sacrifice at the end of the 14-day observation period. During exposure, all animals showed shallow breathing and decreased breathing rate. Shallow breathing occurred from about one hour after initiation of exposure and decreased breathing rate started about half an hour later. The latter symptom generally aggravated during the exposure period. Additionally, most animals were restless, generally at only one time point. About 15 minutes after exposure, all animals showed hunched posture, piloerection and blepharospasm. About two hours later, they additionally showed decreased breathing rate and weakness. From the day after exposure, these symptoms were no longer present, except in the male rat which was found dead on day 2. The surviving animals showed an increased respiratory rate on days 2-5 and one male animal had soft faeces on days 3-5. After day 5, no clinical abnormalities were observed.

Significant weight loss occurred in all animals on the day of exposure and between days 1-3. Between days 3-7, one male and one female lost some more weight whereas the other animals gained weight. During the second week, all survivors showed catch-up growth. At macroscopic examination at the end of the observation period, treatment-related renal and pulmonary changes were seen. The kidneys were enlarged in two male animals. The lungs were affected in one male and all females. Their lungs were pale, had white patches, a few petechiae and, in females only, indentations. In

addition, a large red patch occurred on two lung lobes in one female. As only one animal died following exposure, it was concluded that the 4-hour LC50 of EDTA-CuNa2 in rats was above 5.30 g/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Quality of whole database:

One well performed study, studies with other metal chelates show comparable results (see also section 13 for read across).

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two earlier acute oral toxicity studies were available, one indicating an LD50 value of 890 mg/kg bw, the other indicating an LD50 value of ca. 2100 mg/kg bw. The first study mentioned overnight starvation of the rats, the second study did not mentioning anything on this subject. A RF study prior to the start of an extended OECD 422 study, using a single exposure study in non-starved rats showed no mortality at a level of 1500 mg/kg bw. It was therefore concluded that there was a very large difference in acute toxicity between starved and non-starved rats. However, because the OECD guidelines require testing in overnight starved rats, the LD50 value of 890 mg/kg bw was taken for classification purposes.

Justification for classification or non-classification

EDTA-CuNa2 needs to be classified as GHS Cat. 4, harmful if swallowed. No classification needed for acute inhalation toxicity as the 4 -h LC50 value is in excess of 5.30 g/m3. No classification needed for acute dermal toxicity.