Reaction mass of disodium [2,2'-(imino-κN)dibutanedioato-κ2O1,O4(4-)]cuprum(2-) and sodium sulphate

Endpoint summary

Description of key information

- Acute oral toxicity, OECD 420, rats, LD50 300-2000mg/kg bw
- Acute dermal toxicity, OECD 402, rats, LD50 > 2000 mg/kg bw

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:

2006-12-20 to 2007-01-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Statement of GLP Compliance No. G 024 (Slovak National Accreditation Service); Statement of GLP Compliance No. 4/2006/DPL.

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Institute of Occupational Medicine in Łódź, Poland
- Age at study initiation: Pilot study: 9 week (dose of 2,000 mg/kg bw); 11 week (dose of 300 mg/kg bw). Main experiment: 11 weeks (average body weight of 179 g).
- Weight at study initiation: Pilot study: 168 g (dose of 2,000 mg/kg bw); 198 g (female; dose of 300 mg/kg bw). Main experiment: average body weight of 179 g.
- Fasting period before study: yes. The day before the experiment was due to commence, some 18 hours before administration of the analysed material, the animals were deprived of feed, being left with only water. Feed was made available again 3 hours after administration of the analysed substance.
- Housing: The animals were kept in plastic cages with metal wire covers, with the following dimensions (length x width x height): 58 x 37 x 21 cm.
During the experiment, the animals were kept in cages individually (initial study) and in groups of four (main study).
- Diet (e.g. ad libitum): ad libitum (standard granulated "Murigran" laboratory feed, manufactured by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL of Motycz
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 45-71
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
The analysed substance was administered to the rats on the following days: 20.12.2006. (1 female – initial experiment, dose of 2,000 mg/kg of body mass), 04.01.2007. (1 female – initial experiment, dose of 300 mg/kg of body mass), 09.01.2007. (4 females – experiment proper, dose of 300 mg/kg of body mass). The experiment was terminated on the following days: 20.12.2006. (female that died during the initial experiment, dose of 2,000 mg/kg of body mass), 18.01.2007. (female – initial experiment, dose of 300 mg/kg of body mass), 19.01.2007 (female that died during the experiment proper, dose of 300 mg/kg of body mass), 23.01.2007. (3 females – experiment proper, dose of 300 mg/kg of body mass), respectively.

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 1 ml of the water solution of the analysed substance contained: 400 mg of the substance (dose of 2,000 mg/kg of body mass); 60 mg of the substance (dose of 300 mg/kg of body mass).
- Amount of vehicle (if gavage): 0.5 mL per 100 g of the body weight.

- Rationale for the selection of the starting dose: During the initial experiment, one female received a dose of 2,000 mg/kg of body mass of the analysed substance. Since this female died, the analysed substance was then administered to another female, in a dose of 300 mg/kg of body mass. On the basis of the initial experiment, during the experiment proper the analysed substance was administered to four successive females in a dose of 300 mg/kg of body mass.

Doses:

Initial experiment: 2000 mg/kg bw, 300 mg/kg bw;
Main strudy: 300 mg/kg bw

No. of animals per sex per dose:

Initial experiment: 2000 mg/kg bw (one animal), 300 mg/kg bw (one animal);
Main strudy: four animals

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: An assessment of the general condition of the animals, i.e. observation of all animals in terms of incidence and mortality, was performed twice daily throughout the 14-day duration of the experiment. Detailed clinical observations were performed on the day of administration of the analysed substance (day 0), 10, 30 and 60 minutes after administration, and subsequently every hour over a period of 5 hours from the time of administration. On successive days of the 14-day period of the experiment – once daily.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight.

Preliminary study:

Following the single administration of the analysed substance at the dose of 2,000 mg/kg of body weight to one female (initial experiment), on the same day there were observed changes in posture, the way of walking, kinetic activity and reactivity. The female died within the second hour of administration.
Following the administration of the analysed substance at the dose of 300 mg/kg of body weight to one female (initial experiment), no symptoms of toxicity were observed during the 14-day period of observation. The animal survived the 14-day period of observation.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: based on mortality, clinical signs and findings at necropsy at 2000 mg/kg bw

Mortality:

One female died between the ninth and tenth day of observation. The remaining females survived the 14-day observation period.

Clinical signs:

other: Following the single administration of the analysed substance at the dose of 300 mg/kg bw to four successive females (main experiment), no symptoms of toxicity were observed during the 14-day period of observation for three of the animals. On day seven an

Gross pathology:

The females that died (initial and main studies) and the remaining females that survived following the 14-day period of observation underwent autopsies and macroscopic studies. The macroscopic study of the female that died having received a dose of 2,000 mg/kg bw disclosed hepatic hyperaemia, as well as pale and enlarged lungs. No changes were found during the macroscopic study in the female that died having received a dose of 300 mg/kg bw (the animal was devoured by the other animals present in the cage). Macroscopic studies did not find any pathological changes in the remaining animals.

Table 1. Clinical signs.

Dose(mg/kg of body mass)	Day following administration	Number of live animals	Rat no.
			1*	2	3	4	5
2,000	0 1 2 3 4 5 6 7 8 9 10 11 12 13 14	1 0 0 0 0 0 0 0 0 0 0 0 0 0 0	OK, P - - - - - - - - - - - - - -	- - - - - - - - - - - - - - -	- - - - - - - - - - - - - - -	- - - - - - - - - - - - - - -	- - - - - - - - - - - - - - -
300	0 1 2 3 4 5 6 7 8 9 10 11 12 13 14	5 5 5 5 5 5 5 5 5 5 4 4 4 4 4	BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ	BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ	BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ	BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ BZ	BZ BZ BZ BZ BZ BZ BZ OK OK OK P - - - -

* females from the initial experiment

BZ = without change

OK = clinical symptoms observed

P = died

Table 2. Body weights

Dose mg/kg of body mass	Rat no.	Day of experiment			Difference 14 – 0
		0	7	14
2,000	1*	168	-	-	-
300	1* 2 3 4 5	198 179 174 189 174	233 206 189 219 155	241 197 196 221 -	43 18 22 32 -

* females from the initial experiment

Interpretation of results:

Toxicity Category IV

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the mortality, clinical signs and findings at necropsy in one animal dosed by 2000 mg/kg bw, LD50 is considered to be between 300 - 2000 mg/kg bw that corresponds to Cat. 4 in accordance with the ECHA guidance on the Application of the CLP Criteria (2013).

Executive summary:

A study was conducted to test oral toxicity potential of Cu (II) IDHA in rats. Following the single administration of the analysed substance at dose level of 2000 mg/kg bw to a single female Wistar rat symptoms of toxicity were observed within the first hour of administration. The female died within the second hour of administration of the analysed material. Following the single administration of the analysed substance at dose level of 300 mg/kg bw to a single female, no symptoms of toxicity were observed during the 14-day period of observation. The female survived the 14-day period of observation. Based on this result, the test substance at the dose level of 300 mg/kg bw was administered to four successive females. No symptoms of toxicity were observed during the 14-day period of observation for three of the animals. In one animal, symptoms of toxicity were observed from the seventh day of observation. In all probability, these changes were not connected with the analysed material. The female died on the tenth day of observation. The remaining females survived the 14-day observation period.

The females that died (initial and main studies) and the remaining females that survived the 14-day period of observation underwent autopsies and macroscopic studies.

The macroscopic study of the female that died having received the dose of 2000 mg/kg bw disclosed hepatic hyperaemia, as well as pale and enlarged lungs. No changes were found during the macroscopic study in the female that died having received the dose of 300 mg/kg bw (the animal was devoured by the other animals present in the cage). Macroscopic studies did not find any pathological changes in the remaining animals.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

300 mg/kg bw

Quality of whole database:

The study is conducted in accordance with OECD 420, is GLP compliant and has Klimish score 1.

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:

from 2010-11-10 to 2010-11-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well documented GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SOP/T/21: „Acute dermal toxicity study”

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Slovak National Accreditation Service (No. G-024)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: conventional husbandry of laboratory animals of Institute of Occupational Medicine, Łódź
- Age at study initiation: 10 weeks old
- Weight at study initiation: 295.8 g (males) and 218.2 g (females)
- Fasting period before study: none
- Housing: in cages with dimensions (length x width x height): 58 x 37 x 21 cm; with plastic bottom and wired lid. After application of the test item each animal was kept individually in cage. After removal of test item from animals’ skin during the following days of experiment, the rats were kept five per cage, each separately. UV-sterilized wooden shavings were used as a bedding.
- Diet (e.g. ad libitum): ad libitum standard granulated "Murigran" fodder produced by Wytwórnia Koncentratów i Mieszanek Paszowych AGROPOL, Motycz
- Water (e.g. ad libitum): ad libitum, tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 23 °C;
- Humidity (%): 47 – 63 %
- Air changes (per hr): about 16 times/hour
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: ca. 41 cm² (males) ca. 31 cm² (females)
- Type of wrap if used: gauze patches were covered with PCV foil and elastic bandage was used to make circular protecting band

REMOVAL OF TEST SUBSTANCE
- Washing (if done): water
- Time after start of exposure: 24 hours after application of chemical and immediately after removal of the gauze patch

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2000 mg/kg bw
- Constant volume or concentration used:no
- For solids, paste formed: no (The test item was applied to gauze patches, moistened with few drops of water and then laid on prepared skin.)

VEHICLE
- Amount(s) applied (volume or weight with unit): a few drops of water were applied to the powdered amount of chemical on the gauze patch

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: General and detailed clinical observations were performed in all animals daily during the entire experiment. Body weight of animals was individually determined for each animal: on day 0 (directly before administration of test item) and on 7th and 14th day.
- Necropsy of survivors performed: yes (All animals were sacrificed after 14-day observation period, dissected and subjected to detailed gross necropsy. )
- Other examinations performed: clinical signs, body weight, gross necropsy

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

no mortality occurred

Clinical signs:

other: no general signs of toxicity were stated in the animals. Changes in form of erythema, dryness of epidermis, desquamation of epidermis and scabs were stated on the skin in the site of test item application. Dryness of epidermis and scabs were stated in fiv

Gross pathology:

No pathological changes were stated in animals at gross necropsy.

Other findings:

no other findings reported

Following single application of test item no general signs of toxicity were stated in animals. Changes in form of erythema, dryness of epidermis, desquamation of epidermis and scabs were stated on skin in the site of test item application. Dryness of epidermis and scabs were stated in five males whereas desquamation of epidermis – in one male. Erythema and scabs were stated in four females, dryness of epidermis – in five females, desquamation of epidermis – in two females. These were transient changes except dryness of epidermis in three females which was observed till the end of experiment. All animals survived period of experiment. Slight body weight loss was stated in one female No 2 (6g) and three males No 1 (3g), No 4 (6g) and No 5 (8g) during 1st week of experiment. Body weight gains were stated in all animals after 14-day period of experiment. No pathological changes were stated in animals at gross necropsy.

Table 1: Summary of findings

Cu (II) IDHA: acute dermal toxicity study on rats
Administered dose of test item(mg/kg b.w.)	2000
Sex	males	females
Mortality of animals	0/5	0/5
Clinical signs	no general clinical signs;	no general clinical signs;
	on treated skin: dryness of epidermis and scabs (in 5 males)	on treated skin: erythema and scabs (in 4 females)
	on treated skin: desquamation of epidermis (in 1 male)	on treated skin: dryness of epidermis (in 5 females)
		on treated skin: desquamation of epidermis (in 2 females)

Clinical signs

Following single application of test item no general signs of toxicity were stated in animals. Changes in form of dryness of epidermis from 2nd – 10th to 5th – 13th day and scabs from 2nd to 5th – 9th day after administration were stated in five males on skin in the site of test item application. Furthermore, desquamation of epidermis on 2nd day after administration was stated in one male (No 1). Changes in form of erythema on 2nd day and scabs from 2nd to 9th – 14th day after administration were stated in four females (No 1, No 2, No 3 and No 5). Changes in form of dryness of epidermis from 2nd – 3rd to 4th – 5th day after administration were stated in five females. Furthermore, dryness of epidermis formed in place of scabs was observed in four females (No 1, No 2, No 3 and No 5) from 8th – 9th to 11th – 14th day after administration. Desquamation of epidermis on 2nd day after administration was stated in two females (No 1 and No 4). All animals survived period of experiment. The overall list of results of clinical observations is presented in Table 2.

Table 2 - Clinical signs - overall list
Cu (II) IDHA: acute dermal toxicity study on rats
Dose (mg/kg b.w.)	Sex	Day after administration	Number of alive animals	Rat No
				1	2	3	4	5
2000	males	0	5	NC	NC	NC	NC	NC
		1	5	NC	NC	NC	NC	NC
		2	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		3	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		4	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		5	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		6	5	NC	SIGNS	SIGNS	SIGNS	SIGNS
		7	5	NC	SIGNS	SIGNS	SIGNS	SIGNS
		8	5	NC	SIGNS	SIGNS	SIGNS	SIGNS
		9	5	NC	SIGNS	SIGNS	NC	SIGNS
		10	5	NC	SIGNS	SIGNS	NC	SIGNS
		11	5	NC	SIGNS	SIGNS	NC	SIGNS
		12	5	NC	SIGNS	SIGNS	NC	SIGNS
		13	5	NC	NC	SIGNS	NC	SIGNS
		14	5	NC	NC	NC	NC	NC
	females	0	5	NC	NC	NC	NC	NC
		1	5	NC	NC	NC	NC	NC
		2	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		3	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		4	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		5	5	SIGNS	SIGNS	SIGNS	SIGNS	SIGNS
		6	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		7	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		8	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		9	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		10	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		11	5	SIGNS	SIGNS	SIGNS	NC	SIGNS
		12	5	SIGNS	SIGNS	SIGNS	NC	NC
		13	5	SIGNS	SIGNS	SIGNS	NC	NC
		14	5	SIGNS	SIGNS	SIGNS	NC	NC
NC - no changes
SIGNS - clinical signs

Body weight of animals

Slight body weight loss was stated in one female No 2 (6g) and three males No 1 (3g), No 4 (6g) and No 5 (8g) during 1st week of experiment. Body weight gains were stated in all animals after 14-day period of experiment.

Table 2- Bodyweight of animals (g)-overall list
Cu(II)IDHA:acute dermal toxicity study on rats
Dose(mg/kgb.w.)	Sex	Rat No	Day of experiment/Body weight(g)			Body weight gain(g)(0-14)
			0	7	14
2000	males	1	308	305	310	2
		2	292	295	312	20
		3	299	302	322	23
		4	287	281	296	9
		5	293	285	306	13
	females	1	218	220	237	19
		2	216	210	230	14
		3	219	220	233	14
		4	223	231	235	12
		5	215	228	232	17

Gross examination

At necropsy no pathological changes were stated in males and females sacrificed after 14-day experiment period.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

On the ground of the study, one may state that the median dermal lethal dose (LD50) for Cu (II) IDHA is greater than 2000 mg/kg b.w.

Executive summary:

A study was conducted to test the dermal toxicity potential of Cu (II) IDHA in rats (Kropidlo, A., 2010). Following the single administration of the analysed substance at dose level of 2000 mg/kg bw (semi-occlusive application to the shaved skin for 24 hours) to 5 female and 5 male Wistar rats, no symptoms of general toxicity were observed within the 14 day observation period. No mortality was observed. Changes in form of erythema, dryness of epidermis, desquamation of epidermis and scabs were stated on skin in the site of test item application. Dryness of epidermis and scabs were stated in five males whereas desquamation of epidermis – in one male. Erythema and scabs were stated in four females, dryness of epidermis – in five females, desquamation of epidermis – in two females. These were transient changes except dryness of epidermis in three females which was observed till the end of experiment. Slight body weight loss was stated in one female No 2 (6g) and three males No 1 (3g), No 4 (6g) and No 5 (8g) during 1st week of experiment. Body weight gains were stated in all animals after 14-day period of experiment. No pathological changes were stated in animals at gross necropsy. On the ground of the study, one may state that the median dermal lethal dose (LD50) for Cu (II) IDHA is greater than 2000 mg/kg b.w.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The study is conducted in accordance with OECD 402, is GLP compliant and has Klimish score 1.

Additional information

Acute toxicity: oral

A study was conducted to test oral toxicity potential of Cu(2Na)IDHA in rats (Gruszka, 2007, Report No. OS-46/06, according to OECD 420). Following the single administration of the analysed substance at dose level of 2000 mg/kg bw to a single female Wistar rat symptoms of toxicity were observed within the first hour of administration. The female died within the second hour of administration of the analysed material. Following the single administration of the analysed substance at dose level of 300 mg/kg bw to a single female, no symptoms of toxicity were observed during the 14-day period of observation. The female survived the 14-day period of observation. Based on this result, the test substance at the dose level of 300 mg/kg bw was administered to four successive females. No symptoms of toxicity were observed during the 14-day period of observation for three of the animals. In one animal, symptoms of toxicity were observed from the seventh day of observation. In all probability, these changes were not connected with the analysed material. The female died on the tenth day of observation. The remaining females survived the 14-day observation period.

The females that died (initial and main studies) and the remaining females that survived the 14-day period of observation underwent autopsies and macroscopic studies.

The macroscopic study of the female that died having received the dose of 2000 mg/kg bw disclosed hepatic hyperaemia, as well as pale and enlarged lungs. No changes were found during the macroscopic study in the female that died having received the dose of 300 mg/kg bw (the animal was devoured by the other animals present in the cage). Macroscopic studies did not find any pathological changes in the remaining animals. LD50 was established to be between 300 and 2000 mg/kg bw.

Acute toxicity: dermal

A study was conducted to test the dermal toxicity potential of Cu(2Na)IDHA in rats (Kropidlo, A., 2010, according to OECD 402). Following the single administration of the analysed substance at dose level of 2000 mg/kg bw (semi-occlusive application to the shaved skin for 24 hours) to 5 female and 5 male Wistar rats, no symptoms of general toxicity were observed within the 14 day observation period. No mortality was observed. Changes in form of erythema, dryness of epidermis, desquamation of epidermis and scabs were stated on skin in the site of test item application. Dryness of epidermis and scabs were stated in five males whereas desquamation of epidermis – in one male. Erythema and scabs were stated in four females, dryness of epidermis – in five females, desquamation of epidermis – in two females. These were transient changes except dryness of epidermis in three females which was observed till the end of experiment. Slight body weight loss was stated in one female No 2 (6g) and three males No 1 (3g), No 4 (6g) and No 5 (8g) during 1st week of experiment. Body weight gains were stated in all animals after 14-day period of experiment. No pathological changes were stated in animals at gross necropsy. LD50 was considered to be greater than 2000 mg/kg bw.

Justification for selection of acute toxicity – oral endpoint
Only one study is available

Justification for selection of acute toxicity – dermal endpoint
Only one study is available

Justification for classification or non-classification

Based on the LD50 of 300 - 2000 mg/kg bw established in the acute oral toxicity study, Cu(2Na)IDHA is subject to classification and labelling for acute toxic effects by oral route of exposure in accordance with European Regulation (EC) No. 1272/2008 (Cat 4; H302 Harmful if swallowed).

Based on LD50 of greater than 2000 mg/kg bw established in the dermal acute toxicity study, Cu(2Na)IDHA is not subject to classification and labelling for acute toxic effects by dermal route of exposure in accordance with European Regulation (EC) No. 1272/2008.