Dicopper hydroxide phosphate

Endpoint summary

Description of key information

Key studies exist for all three acute toxicity endpoints; these studies are conducted in accordance with appropriate guidelines and under the conditions of GLP. As such these data are considered to be adequate and reliable for use as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

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:

Study was performed between 23/11/2010 and 07/12/2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

The test species used in this study was mice. The test guideline states that the preferred rodent species is rat; however the guideline also states that other rodent species may also be used and as such this is not considered to be a deficiency.

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): dicopper hydroxide phosphate
-CAS Number: 12158-74-6
- EC Number: 235-285-2
- Purity test date: 15/01/2009
- Lot/batch No.: 08004
- solid: particulate/powder

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BIOSERV
- Age at study initiation: no data
- Weight at study initiation: 17.8 g - 21.2 g
- Fasting period before study: 3 hours
- Housing: no data
- Diet (e.g. ad libitum): ad libitum, no diet 3 hours before and 1 hour after application
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23°C
- Humidity (%): 40 - 60%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Test solution and vehicle (distilled water were administered by force-feeding under a volume of 2 ml/100 g bodyweight, using a suitable graduated syringe fitted with an oesophageal metal cannula.

Doses:

300 mg/kg bw
2000 mg/kg bw

No. of animals per sex per dose:

3 females per dose.
6 females in control groups.

Control animals:

yes

Remarks:

All control animal recieved the vehicle (distilled water).

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: weighed on Day 0, Day 7 and Day 14.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs and body weight

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

2 animals treated with 2000 mg/kg bodyweight died between day 1 and day 14.
None of the animals treated with 300 mg/kg bodyweight died during the 14 day observation period.

Clinical signs:

other: See tables below.

Gross pathology:

See tables below.

Test series 1 – Animals treated with 2000 mg/kg bw

 Table 1: Clinical observations from T0 to 4 hours

Observation	Test group 1 (2000 mg/kg bw)			Control group 1 (vehicle
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
Spontaneous activity	NAD	NAD	NAD	NAD	NAD	NAD
Preyer’s reflex (noise)	NAD	NAD	NAD	NAD	NAD	NAD
Respiratory rate	NAD	NAD	NAD	NAD	NAD	NAD
Convulsions	NAD	NAD	NAD	NAD	NAD	NAD
Tremors	NAD	NAD	NAD	NAD	NAD	NAD
Body temperature (evaluated by touch)	NAD	NAD	NAD	NAD	NAD	NAD
Muscle tone	NAD	NAD	NAD	NAD	NAD	NAD
Palpebral opening	NAD	NAD	NAD	NAD	NAD	NAD
Pupil appearance	NAD	NAD	NAD	NAD	NAD	NAD
Salivation	NAD	NAD	NAD	NAD	NAD	NAD
Lachrymation	NAD	NAD	NAD	NAD	NAD	NAD
Righting reflex	NAD	NAD	NAD	NAD	NAD	NAD
Back hair appearance	NAD	NAD	NAD	NAD	NAD	NAD
MORTALITY	0	0	0	0	0	0

 NAD = No abnormalities noted

  

Table 2: Clinical observations from day 1 to day 14

Observation	Test group 1 (2000 mg/kg bw)			Control group 1 (vehicle)
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
Spontaneous activity	Decreased	Decreased	NAD	NAD	NAD	NAD
Preyer’s reflex (noise)	NAD	NAD	NAD	NAD	NAD	NAD
Respiratory rate	NAD	NAD	NAD	NAD	NAD	NAD
Convulsions	NAD	NAD	NAD	NAD	NAD	NAD
Tremors	NAD	NAD	NAD	NAD	NAD	NAD
Body temperature (evaluated by touch)	Decreased	Decreased	NAD	NAD	NAD	NAD
Muscle tone	Decreased	Decreased	NAD	NAD	NAD	NAD
Palpebral opening	NAD	NAD	NAD	NAD	NAD	NAD
Pupil appearance	NAD	NAD	NAD	NAD	NAD	NAD
Salivation	NAD	NAD	NAD	NAD	NAD	NAD
Lachrymation	NAD	NAD	NAD	NAD	NAD	NAD
Righting reflex	Limited	Limited	NAD	NAD	NAD	NAD
Back hair appearance	NAD	NAD	NAD	NAD	NAD	NAD
MORTALITY	1	1	0	0	0	0

 NAD = No abnormalities noted

 

Table 3: body weight and weight gain in grams

Day	Test group 1 (2000 mg/kg bw)			Control group 1 (vehicle)
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
Day 0	21.2	20.6	20.5	18.6	19.4	17.8
Day 7	n/a	n/a	17.4	20.3	20.2	21.0
Day 7 – Day 0	n/a	n/a	-3.1	1.7	0.8	3.2
Day 14	n/a	n/a	17.1	20.8	21.0	20.9
Day 14 – Day 0	n/a	n/a	-3.4	2.2	1.6	3.1

 n/a = not applicable due to early mortality.

 

Table 4: macroscopic observation at necropsy

Observed Organs	Test group 1 (2000 mg/kg bw)			Control group 1 (vehicle)
	Animal 1	Animal 2	Animal 3	Animal 1	Animal 2	Animal 3
Oesophagus	NAD	NAD	NAD	NAD	NAD	NAD
Stomach	1, 2	1, 3	NAD	NAD	NAD	NAD
Duodenum	NAD	NAD	NAD	NAD	NAD	NAD
Jejunum	NAD	NAD	NAD	NAD	NAD	NAD
Ileum	NAD	NAD	NAD	NAD	NAD	NAD
Caecum	NAD	NAD	NAD	NAD	NAD	NAD
Colon	NAD	NAD	NAD	NAD	NAD	NAD
Rectum	NAD	NAD	NAD	NAD	NAD	NAD
Spleen	NAD	NAD	NAD	NAD	NAD	NAD
Liver	NAD	NAD	NAD	NAD	NAD	NAD
Thymus	NAD	NAD	NAD	NAD	NAD	NAD
Trachea	NAD	NAD	NAD	NAD	NAD	NAD
Lungs	NAD	NAD	NAD	NAD	NAD	NAD
Heart	NAD	NAD	NAD	NAD	NAD	NAD
Kidneys	NAD	NAD	4	NAD	NAD	NAD
Urinary bladder	NAD	NAD	NAD	NAD	NAD	NAD
Ovaries	NAD	NAD	NAD	NAD	NAD	NAD
Uterus	NAD	NAD	NAD	NAD	NAD	NAD
Adrenals	NAD	NAD	NAD	NAD	NAD	NAD
Pancreas	NAD	NAD	NAD	NAD	NAD	NAD

 

NAD = No abnormality detected

1.    Strong gas development

2.    Test material still visible in stomach 2 days after application

3.    Test material still visible in stomach 3 days after application

4.    Atypical appearance of kidneys (yellow) due to restricted blood flow

[SEE 'OVERALL REMARKS, ATTACHMENTS' FOR DATA RELATING TO ANIMALS TESTED WITH 300 MG/KG BW]

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 of dicopper hydroxide phosphate has been determined to be in the range of > 300 to < 2000 mg/kg bw.

Executive summary:

The LD50 of dicopper hydroxide phosphate has been determined to be in the range of > 300 to < 2000 mg/kg bw and as such is classified as acutely toxic via the oral route, category 4 (EU CLP).  This study has been selected as the key study in accordance with Regulation (EC) No. 1907/2006 (REACH) because the results are sufficient in order to derive a reliable conclusion on classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Quality of whole database:

LD50 was found to be in the range: >300 - <2000 mg/kg bw
The study is conducted under the conditions of GLP and in accordance with an appropriate guideline (OECD 423). This study has been assigned a Klimisch reliability of 1. The database is considered to be of high quality.

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:

The study was performed between 16 April 2012 and 14 May 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:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Date of Inspection: 19-21 July 2011, Date of signature: 31 August 2011

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Sponsor's identification: Dicopper hydroxide phosphate
Description: Pale green powder
Batch number: MV 500
Purity: Neat Substance
Date received: 14 March 2012
Expiry Date: Not supplied
Storage conditions: Room temperature, in the dark

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Further details on strain: RccHanTM : WIST strain rats
- Source: Harlan Laboratories UK Ltd, Oxon, UK
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 200-350 g
- Fasting period before study: none
- Housing: The animals were housed in groups of up to three by sex in solid-floor polypropylene cages with stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK) and provided with environmental enrichment items: wooden chew blocks and cardboard “fun tunnels” (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): with the exception of the exposure period diet (Harlan 2014C Rodent Diet, Harlan Laboratories UK Ltd, Oxon, UK) was available ad libitum
- Water (e.g. ad libitum): with the exception of the exposure period water was available ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: A dust atmosphere was produced from the test item using a SAG 410 Solid Aerosol Generator (TOPAS GmbH, Dresden, Germany) located adjacent to the exposure chamber. The SAG 410 was connected to a metered compressed air supply.
Compressed air was supplied by means of an oil free compressor and passed through a water trap and respiratory quality filters before it was introduced to the SAG 410.
- Exposure chamber volume: 30 litres (dimensions: 28 cm diameter x 50 cm high)
-Chamber flow rate: The chamber flow rate was maintained at 60 L/min providing 120 air changes per hour.
- Method of holding animals in test chamber: each rat was individually held in a tapered, polycarbonate restraining tube fitted onto a single tier of the exposure chamber and sealed by means of a rubber ‘O’ ring. Only the nose of each animal was exposed to the test atmosphere.
- Method of conditioning air: The concentration within the chamber was controlled by adjusting the test item feed rate from the SAG 410. The extract from the exposure chamber passed through a ‘scrubber’ trap and was connected with a high efficiency filter to a metered exhaust system. The chamber was maintained under negative pressure.
- System of generating particulates/aerosols: In order to facilitate aerosolisation and reduce particle size, the test item was ground using a small amount of diethyl ether in a Retsch Planetary Ball Mill (Retsch (UK) Ltd, Leeds, UK) all of the solvent was removed via evaporation prior to use. The absorption of the test item was not determined. See 'exposure apparatus' for further details.
- Method of particle size determination: The particle size of the generated atmosphere inside the exposure chamber was determined three times during the exposure period using a Marple Personal Cascade Impactor (Westech IS Ltd, Beds., UK). This device consisted of six impactor stages (9.7, 6.7, 3.8, 1.8, 0.94 and 0.46 μm cut points) with stainless steel collection substrates and a back up glass fibre filter, housed in an aluminium sampler. The sampler was temporarily sealed in a sampling port in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through it using a vacuum pump.
The collection substrates and backup filter were weighed before and after sampling and the weight of test item, collected at each stage, calculated by difference.
The mean amount for each stage was used to determine the cumulative amount below each cut-off point size. In this way, the proportion (%) of aerosol less than 9.7, 6.7, 3.8, 1.8, 0.94 and 0.46 μm was calculated.
The resulting values were converted to probits and plotted against Log10 cut-point size. From this plot, the Mass Median Aerodynamic Diameter (MMAD) was determined (as the 50% point) and the geometric standard deviation was calculated. In addition the proportion (%) of aerosol less than 4 μm (considered to be the inhalable fraction) was determined.
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity inside the exposure chamber were measured by an electronic thermometer/humidity meter (Hanna Instruments Ltd, Beds., UK) located in a vacant port in the animals’ breathing zone of the chamber and recorded every thirty minutes throughout the four-hour exposure period. Individual values are given in Appendix 9 (attached).
-Exposure chamber oxygen concentration: Oxygen levels within the exposure chamber were measured by an electronic oxygen analyser (Servomex (UK) Ltd, Crowborough, East Sussex) located in a port in the animals breathing zone during the four-hour exposure period. The test atmosphere was generated to contain at least 19% oxygen. Individual values are given in Apenndix 9 (attached).

TEST ATMOSPHERE
- Brief description of analytical method used: Homogeneity of the test atmosphere within the chamber was not specifically determined during this study. Chambers of the same design (ADG Developments Ltd, Hitchin, Herts, UK) have been fully validated and shown to produce evenly distributed atmospheres in the animals’ breathing zone with a wide variety of test items (Green J D et al, 1984).
Prior to the start of the study, test item atmospheres were generated within the exposure chamber. During this characterisation period test item input rates, grinding techniques and generation systems were varied in order to achieve the required atmospheric conditions.
- Samples taken from breathing zone: yes
The actual chamber concentration was measured at regular intervals during the exposure period. The gravimetric method used glass fibre filters placed in a filter holder. The holder was temporarily sealed in a vacant port in the exposure chamber in the animals’ breathing zone and a suitable, known volume of exposure chamber air was drawn through the filter using a vacuum pump.
Each filter was weighed before and after sampling in order to calculate the weight of collected test item. The difference in the two weights, divided by the volume of atmosphere sampled, gave the actual chamber concentration.
The nominal chamber concentration was calculated by dividing the mass of test item used by the total volume of air passed through the chamber.
The nominal concentration is 240% of the actual mean achieved atmosphere concentration and shows that keeping the aerosol airborne was moderately difficult.

Analytical verification of test atmosphere concentrations:

no

Duration of exposure:

4 h

Concentrations:

5.09 mg/L (mean achieved concentration)

No. of animals per sex per dose:

3 males, 3 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All animals were observed for clinical signs at hourly intervals during exposure, immediately on removal from the restraining tubes at the end of exposure, one hour after termination of exposure and subsequently once daily for up to fourteen days. Any evidence of overt toxicity was recorded at each observation.
- Bodyweight: Individual bodyweights were recorded on arrival, prior to treatment on the day of exposure and on Days 1, 3, 7 and 14.
- Necropsy of survivors performed: yes
At the end of the fourteen day observation period the animals were killed by intravenous overdose of sodium pentobarbitone. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded. The respiratory tract was subjected to a detailed macroscopic examination for signs of irritancy or local toxicity.

Statistics:

Evaluation of Data
Data evaluations included the relationship, if any, between the animals’ exposure to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, necropsy findings, bodyweight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute inhalation median lethal concentration (LC50) of the test item was made.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.09 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

There was no mortality. Individual data are given in Appenix 3 (attached)

Clinical signs:

other: Individual clinical observations are given in Appendices 4 and 5. Signs of hunched posture and pilo-erection are commonly seen in animals for short periods on removal from the chamber following 4-Hour inhalation studies. Wet fur is commonly recorded both

Body weight:

Individual bodyweights, together with bodyweight changes, are given in Appendix 6.
All animals exhibited bodyweight losses on the first day post-exposure and from Days 1 to 3 post-exposure, with the exception of one male animal which exhibited a bodyweight gain from Days 1 to 3. All animals exhibited bodyweight gains during the remainder of the recovery period.

Gross pathology:

Individual necropsy findings are given in Appendix 7.
All animals exhibited enlarged lungs that were abnormally dark or had dark patches at necropsy.

Interpretation of results:

GHS criteria not met

Conclusions:

It was considered that the acute inhalation median lethal concentration (4 hr LC50) of Dicopper hydroxide phosphate, in the RccHanTM : WIST strain rat, was greater than 5.09 mg/L.

Executive summary:

No deaths occurred in a group of six rats exposed to a mean achieved atmosphere concentration of 5.09 mg/L for four hours. It was therefore considered that the acute inhalation median lethal concentration (4 hr LC50) of Dicopper hydroxide phosphate, in the RccHanTM : WIST strain rat, was greater than 5.09 mg/L  (EU CLP – unclassified).

This study is considered to be acceptable for use as a key study in accordance with Regulation (EC) No. 1907/2006 (REACH) and for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP). Further testing is scientifically unjustified.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating conc.

Value:

5 090 mg/m³ air

Quality of whole database:

LC50: >5090 mg/m3
The study is conducted under the conditions of GLP and in accordance with an appropriate guideline (OECD 436). This study has been assigned a Klimisch reliability of 1. The database is considered to be of high quality.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The test was performed between 28/06/2011 and 13/07/2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): dicopper hydroxide phosphate
- Batch number: 08004
- CAS number: 12158-74-6
- EC number: 235-285-2
- solid: particulate/powder

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Deutschland
- Age at study initiation: Guinea pigs were young adults at the start of the study.
- Weight at study initiation: 350 - 450 g
- Housing: Guinea-pigs were kept in separate cages.
- Diet (e.g. ad libitum): Commercial feeding mixture was fed ad libitum.
- Water (e.g. ad libitum): Water was supplemented with 1g/l vitamin C. Access was ad libitum.
- Acclimation period: The animals were acclimated and permanently controlled by veterinarians according to DIN ISO 10993-2.

Type of coverage:

semiocclusive

Vehicle:

water

Details on dermal exposure:

TEST SITE
- Area of exposure: The fur was removed from the dorsal area of the trunk of the test animals 24 hours before the test.Test material was applied over an area which is approximately 10% of the total body surface.
- % coverage: 10%
- Type of wrap if used: Test substance was held in contact with the skin with gauze dressing and tape throughout a 24 hour exposure period.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Not done.


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The animals recieved an effective dose of 2000 mg/kg bodyweight moistened with water to ensure good skin contact.

Duration of exposure:

24 hours.

Doses:

2000 mg/kg bodyweight

No. of animals per sex per dose:

5 males and 5 females.

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The test animals were observed frequently during the first day and then at least once each day. The weighing of the test animals was carried out shortly before the test substance was applied and weekly thereafter.
- Necropsy of survivors performed: yes - a necropsy of all animals was carried out after 14 days.
- Other examinations performed:body weight

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality was observed.

Clinical signs:

other: No test animals exhibited adverse physical symptoms or signs of toxicity after the application of the test material over the observation period of 14 days.

Gross pathology:

A through gross pathological examination was performed by a pathologist. No abnormalities were noted in any of the animals.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute dermal LD50 was determined to be > 2000 mg/kg bw.

Executive summary:

The test material did not induce toxic reactions in any of the test animals after dermal application and within an observation period of 14 days.

This study is considered to be acceptable and to adequately satisfy both the guideline requirement and the regulatory requirement (Regulation (EC) No. 1907/2006; REACH) as a key study for this endpoint. In addition, this study is considered to be acceptable for classification and labelling in accordance with Regulation (EC) No. 1272/2008 (EU CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Quality of whole database:

LD50: >2000 mg/kg bw

The study is conducted under the conditions of GLP and in accordance with an appropriate guideline (OECD 406). This study has been assigned a Klimisch reliability of 1. The database is considered to be of high quality.

Justification for classification or non-classification

Acute oral toxicity: The oral LD₅₀ has been determined to be within the range 300 - 2000 mg/kg bw and therefore in accordance with Regulation (EC) No. 1272/2008 (EU CLP) dicopper hydroxide phosphate is classified as acutely toxic via the oral route category 4.

Acute inhalation toxicity: The LC₅₀ has been determined to be >5.09 mg/L and therefore in accordance with Regulation (EC) No. 1272/2008 (EU CLP) dicopper hydroxide phosphate is not considered to be classified as acutely toxic via the inhalation route.

 

Acute dermal toxicity: The dermal LD50 has been determined to be >2000 mg/kg bw and therefore in accordance with Regulation (EC) No. 1272/2008 (EU CLP) dicopper hydroxide phosphate is not considered to be classified as acutely toxic via the dermal route.

No further testing is considered to be necessary.