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Toxicological information

Acute Toxicity: inhalation

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Administrative data

Endpoint:
acute toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
August 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed and reported GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name: EDTA-CuNa2
Chemical name: Ethylenediaminetetraacetic acid, copper disodium complex
CAS registry number: 14025-15-1
Appearance: blue microgranules
Molecular formula: C10H12N2O8CuNa2
Molecular weight: 397.7 g/mol g/mol
Batch number: CFC-10334 (304S0001)
Purity: 92.7%
Storage conditions: ambient temperature (15-25°C), protected from moisture
Expiry date: February 2013
Date of receipt: 6 December 2011

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals 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.

Administration / exposure

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)

Results and discussion

Effect levels
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 5.32 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

Applicant's summary and conclusion

Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
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.