copper sulphide and zinc chloride sulphate hydroxide, recovery products from copper and zinc dusts

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic (Ames).

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 March 2020 - 05 June 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name Sulphide Precipitate
Batch no. 20190601
CAS no. unknown
Composition 2.9 % Cu; 18.0 % Zn; 0.5 % Fe; 0.1 % Pb; 0.7 % Cd; 0.4 % As; 0.3 % Sb; 0.1 % Sn; 0.0 % Bi; 0.0 % Br; 2.7 % Cl; 1.1 % F
Storage room temperature (20 ± 5 °C)
Expiry date 31. Dec. 2025
Stability stable under storage conditions
Appearance blackish-green, wet sticky solid
Purity 100 % (UVCB)
Homogeneity homogeneous

Production date not stated
EC no. unknown
Molecular formula not stated
Molecular weight not stated
Vapour pressure not stated
Solubility in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated
Stability in solvents H2O: not stated; EtOH: not stated; acetone: not stated; CH3CN: not stated; DMSO: not stated


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized (demin.) water, dimethyl sulfoxide (DMSO), acetone and ethanol.
The test item is not completely soluble in a concentration of 50 g/L in any of the solvents.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bac-teria or the number of spontaneous revertants in the tested concentrations.
Therefore, for every test item concentration the test item was weighed directly and every suspension was prepared separately, starting with the highest solution containing 50 ± 5 g/L suspended in DMSO. The suspensions were stirred during the experiment.

The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.

The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313 and 156 µg/plate.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

CELLS USED
All Salmonella typhimurium strains were obtained from Trinova BioChem GmbH (batch: TA98: 5511D, TA100: 5343D, TA102: 5405D, TA1535: 5416D, TA1537:5428D) and were stored as lyophilizates in the refrigerator at 2 – 8 °C.

On the day before the start of each experiment, a nutrient broth (Oxoid nutrient broth no. 2) was inoculated with one lyophilizate per strain at 4:00 pm.
These overnight cultures were placed in the heating chamber at 37 ± 1 °C for 16 hours.
For the last two hours the overnight cultures were shaken on an orbital shaker (150 rpm) at 37 ± 1 °C.
Afterwards, the overnight cultures were ready for use in the experiment.
During the test, the overnight cultures were stored at room temperature (20 ± 5 °C) as to prevent changes in the titre.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S9)

Test concentrations with justification for top dose:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized (demin.) water, dimethyl sulfoxide (DMSO), acetone and ethanol.
The test item is not completely soluble in a concentration of 50 g/L in any of the solvents.
Based on the non-GLP pre-test, DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bac-teria or the number of spontaneous revertants in the tested concentrations.
Therefore, for every test item concentration the test item was weighed directly and every suspension was prepared separately, starting with the highest solution containing 50 ± 5 g/L suspended in DMSO. The suspensions were stirred during the experiment.

The following nominal test item concentrations were prepared for experiment 1:
5000, 1500, 500, 150 and 50 µg/plate.

The following nominal test item concentrations were prepared for experiment 2:
5000, 2500, 1250, 625, 313 and 156 µg/plate.

Vehicle / solvent:

The vehicle of the test item was Dimethylsulfoxide (DMSO), CAS No. 67-68-5,
batch: 2018270919

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO; demineralised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

benzo(a)pyrene

other: 2-Amino-Anthracene

Details on test system and experimental conditions:

On the day of the test, the bacteria cultures were checked for growth visually. The incuba-tion chambers were heated to 37 ± 1 °C. The water bath was turned to 43 ± 1 °C. The table surface was disinfected.
The S9-mix was freshly prepared and stored at 0 °C.

Experiment 1
Date of treatment: 26. May 2020
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5000, 1500, 500, 150, 50 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: plate incorporation method


Experiment 2
Date of treatment: 03. Jun. 2020
Tested strains: TA98, TA100, TA102, TA1535, TA1537
Test item concentrations: 5000, 2500, 1250, 625, 313, 156 µg/plate
Incubation time: 48 h
Incubation temperature: 37 ± 1 °C
Method: pre-incubation method

Description of the Method
Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used.
The test item suspensions were prepared according to chapter 6.1.3, page 11.
For the top agar 100 mL agar basis was melted in a microwave oven, 10 mL of the histi-dine-biotin-solution 0.5 mM was added, then the mixture was placed in the water bath at 43 ± 1 °C.

Plate incorporation method
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:

• 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
• 500 µL S9-mix (see chapter 6.4.13, page 17 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
• 2000 µL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark incubator at 37 ± 1 °C.

Pre-incubation method
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:

• 100 µL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
• 500 µL S9-mix (see chapter 6.4.13, page 17 for test with metabolic activation) or phos-phate buffer (for test without metabolic activation).
• 100 µL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)

After the pre-incubation for 20 minutes, 2000 µL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.
 

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

Five different analysable and non-toxic concentrations should be used for the evaluation of the mutagenic potential of the test item.
A result is considered as positive if a clear and dose-related increase in the number of revertants occurs and/or a biologically relevant positive response for at least one of the concentrations occurs in at least one tester strain with or without metabolic activation.

A biologically relevant increase is described as follows:
• if in the bacteria strains TA 98, TA 100 and TA 102 the number of revertants is at least twice as high than the reversion rate of the negative controls (increase factor of at least 2)
• if in the bacteria strains TA 1535 and TA 1537 the number of revertants is at least three times higher than the reversion rate of the negative controls (increase factor of at least 3).

A substance is not mutagenic if it does not meet the criteria above.
If the criteria listed above are not clearly met, the results are assessed as equivocal and will be discussed.
Tables and, if applicable, graphs of the values are included in this final report. It is stated, at which concentration (100% test item extract/plate) mutagenicity could be observed. The lowest concentration which showed mutagenicity is decisive of the assessment of the test item.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Mean Revertants Experiment 1

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	13	14	58	58	261	261	7	5	5	6
	sd	1.5	5.5	6.5	7.5	18.5	12.2	2.0	1.7	1.0	0.6
DMSO	Mean	12	14	52	60	267	285	6	6	6	4
	sd	1.5	1.5	5.7	5.7	16.7	16.7	2.1	1.0	1.0	1.2
Positive Controls*	Mean	s.g.	113	576	s.g.	619	768	347	123	83	161
	sd	--	10.1	32.0	--	20.1	36.7	9.2	9.0	5.6	6.1
	f(I)	> 2	8.07	9.93	> 2	2.32	2.69	49.57	20.50	13.83	40.25
5000 µg/plate	Mean	11	16	69	63	267	264	6	5	4	6
	sd	3.2	1.0	12.9	9.5	4.6	8.0	0.6	2.0	0.6	0.6
	f(I)	0.92	1.14	1.33	1.05	1.00	0.93	1.00	0.83	0.67	1.50
1500 µg/plate	Mean	12	16	62	51	269	272	7	4	4	4
	sd	2.6	3.1	4.6	7.0	4.6	8.0	2.0	1.0	0.6	0.6
	f(I)	1.00	1.14	1.19	0.85	1.01	0.95	1.17	0.67	0.67	1.00
500 µg/plate	Mean	14	17	60	52	267	277	6	7	4	6
	sd	2.5	4.0	10.0	1.5	9.2	4.6	2.0	0.6	0.6	0.6
	f(I)	1.17	1.21	1.15	0.87	1.00	0.97	1.00	1.17	0.67	1.50
150 µg/plate	Mean	9	22	64	57	261	277	5	6	4	5
	sd	2.5	1.2	6.7	7.5	12.2	4.6	1.0	2.1	0.6	1.0
	f(I)	0.75	1.57	1.23	0.95	0.98	0.97	0.83	1.00	0.67	1.25
50 µg/plate	Mean	11	15	57	65	280	272	9	6	4	5
	sd	2.1	0.6	4.6	8.4	8.0	8.0	0.6	1.2	0.0	1.2
	f(I)	0.92	1.07	1.10	1.08	1.05	0.95	1.50	1.00	0.67	1.25

Mean Revertants Experiment 2

Strain		TA98		TA100		TA102		TA1535		TA1537
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	12	12	75	53	299	325	7	8	5	7
	sd	1.7	1.2	1.0	1.5	25.7	28.1	1.0	1.7	1.2	1.7
DMSO	Mean	11	14	58	59	333	333	8	7	4	6
	sd	3.8	4.4	7.1	7.0	16.7	12.2	1.2	0.6	0.0	1.5
Positive Controls*	Mean	s.g.	48	531	s.g.	725	s.g.	177	168	71	123
	sd	--	4.5	20.1	--	12.2	--	4.6	18.3	3.2	2.3
	f(I)	> 2	3.43	7.08	> 2	2.18	> 2	25.29	24.00	17.75	20.50
5000 µg/plate	Mean	11	13	87	77	296	333	9	9	6	5
	sd	1.5	1.0	7.6	2.1	8.0	9.2	0.6	0.0	1.0	0.0
	f(I)	1.00	0.93	1.50	1.31	0.89	1.00	1.13	1.29	1.50	0.83
2500 µg/plate	Mean	9	13	83	79	296	323	10	10	4	5
	sd	1.5	1.2	13.7	3.6	8.0	12.2	0.6	1.5	0.6	0.6
	f(I)	0.82	0.93	1.43	1.34	0.89	0.97	1.25	1.43	1.00	0.83
1250 µg/plate	Mean	10	16	72	68	312	341	8	8	5	5
	sd	3.2	2.6	9.0	12.7	13.9	12.2	2.3	0.0	0.0	0.6
	f(I)	0.91	1.14	1.24	1.15	0.94	1.02	1.00	1.14	1.25	0.83
625 µg/plate	Mean	9	20	74	67	352	320	8	9	5	6
	sd	3.1	3.5	6.0	2.5	8.0	8.0	1.0	1.2	1.0	0.6
	f(I)	0.82	1.43	1.28	1.14	1.06	0.96	1.00	1.29	1.25	1.00
313 µg/plate	Mean	14	16	73	65	331	333	9	10	5	5
	sd	0.6	4.6	5.1	6.6	12.2	12.2	0.0	1.7	1.2	1.5
	f(I)	1.27	1.14	1.26	1.10	0.99	1.00	1.13	1.43	1.25	0.83
156 µg/plate	Mean	12	17	60	61	344	341	10	10	5	5
	sd	3.2	3.8	2.3	7.8	13.9	4.6	0.6	1.2	1.0	0.6
	f(I)	1.09	1.21	1.03	1.03	1.03	1.02	1.25	1.43	1.25	0.83

Conclusions:

Based on the results of this study it is concluded that Sulphide precipitate is not mutagen-ic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study.

Executive summary:

Two valid experiments were performed.

 

The study procedures described in this report were based on the most recent Guideline OECD 471 and EU Method B.13/14.

The test item Sulphide precipitate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

 

Experiment 1:

In experiment 1, the test item (suspended DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

In experiment 1, the test item (suspended DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two valid experiments were performed.

The study procedures described in this report were based on the most recent Guideline OECD 471 and EU Method B.13/14.

The test item Sulphide precipitate was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).

The test was performed in two experiments in the presence and absence of metabolic activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing for absence of metabolic activation.

 

Experiment 1:

In experiment 1, the test item (suspended DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2:

In experiment 1, the test item (suspended DMSO) was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the presence and the absence of metabolic activation.

Justification for classification or non-classification

The substance is not classified for mutagenic according to the CLP Regulation No. 1272/2008.