Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available genotoxicity studies with ZPS and the knowledge on the genotoxicity profile of zinc, ZPS is not considered genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
aroclor-1254 induced rat liver
Test concentrations with justification for top dose:
Without metabolic activation: 1.0, 8.0, 15, 22, 29, 36, 43 and 50 µL/mL
With metabolic activation: 0.02, 0.16, 0.30, 0.44, 0.58, 0.72, 0.86 and 1.0 µL/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: FoP (indicated by sponsor as preferred solvent)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation
Details on test system and experimental conditions:
DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 20 h and 44 h at 37°C

SELECTION AGENT (mutation assays): trifluorothymidine

VIABILITY PARAMETERS
(relative) suspension growth, relative cloning efficiency, total growth
Evaluation criteria:
Criteria for a negative response
- All of the cultures exhibiting a total growth of 10% and greater have mutant frequencies that are less than twice that of the mean mutant frequency of the corresponding solvent control cultures
- There is no evidence of a dose-dependent response

Criteria for a positive response
- A response is considered positive if at least one culture has a mutant frequency that is two times or more greater than the average mutation frequency of the corresponding solvent control cultures and the response is dose dependent

Criteria for an equivocal response
- A response is considered equivocal if it does not fulfill the criteria of either a negative or a positive response, and/or the Study Director does not consider theresponse to be either positive or negative
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the presence and absence of metabolic activation, the substance induced a significant increase in the mutant frequency of treated cultures as compared to the mutant frequency of the corresponding solvent control cultures. The response was dose dependent.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
other information
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Metabolic activation system:
aroclor induced rat liver microsomes
Test concentrations with justification for top dose:
0.2, 1.0, 5.0, 10 and 20 µL/plate
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
strains TA98, TA1538 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
strains TA100, TA1535 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 9-aminoacridine
Remarks:
strain TA1537 without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
strains TA98, TA100, TA1535, TA1537, TA1538 with metabolic activation
Details on test system and experimental conditions:
Study type: plate incorporation assay
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Strain Target gene
TA 1537 his C 3076
TA 98 his D 3052
TA 1535 his G 46
TA 100 his G 46
WP2 uvrA Trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/p-Naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
33, 100, 333, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
Deionised water
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Strains TA1535 and TA100, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Strains TA1537 and TA98, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Strain WP2 uvrA, without metabolic activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
All strains, with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

EXPERIMENT 1
DURATION
- Preincubation period: -
- Exposure duration: 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth / reduction in the number of revertants

EXPERIMENT 2
DURATION
- Preincubation period: 60 minutes
- Exposure duration: 72 hours at 37°C in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth / reduction in the number of revertants
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
No statistical evaluation of the data is required.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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
Additional information on results:
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.
No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
GLP compliance:
yes
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Species / strain / cell type:
hepatocytes: Sprague-Dawley rat
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.003, 0.01, 0.03, 0.1, 0.3, 0.6, 1.0, 2.0 and 3.0 µL/mL (a full evaluation was at five concentrations from 0.01 µL/mL to 0.6 µL/mL)
Vehicle / solvent:
Williams Medium E
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18-20 h

NUMBER OF REPLICATIONS: 3 (UDS) and 2 (cytotoxicity)

FIXATION AGENT
ethanol-acetic acid fixative

NUMBER OF CELLS EVALUATED: 25

DETERMINATION OF CYTOTOXICITY
- Method: Trypan blue exclusion method / visual inspection
Evaluation criteria:
If the mean net nuclear count was increased by at least five counts over the control, the results for a particular dose level were considered significant. A test article was judged positive if it induced a dose-related response and at least one dose produced a significant increase in the average net nuclear grains when compared to that of the control. In the absence of the dose response, a test article which showed a significant increase in the mean net nuclear grain count in at least two successive doses was considered positive. If a test article showed a significant increase in the net nuclear grain count at one dose level without any dose response, the test article was considered to have a marginal positive activity. The test article was considered negative if no significant increase in the net nuclear grain counts at any dose level was observed.
Statistics:
Means, standard deviations and percent survivals were computed using LOTUS 1-2-3 program.
Species / strain:
hepatocytes: Sprague-Dawley rat
Metabolic activation:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 0.3 µL/mL and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Dose levels 0.01 to 0.6 µL/mL were evaluated, dose levels 1.0 - 3.0 µL/mL caused too much toxicity to evaluate the nuclei for UDS.
None of the substance concentrations caused a significant increase in the mean net nuclear counts.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07-31-2020 - 04-16-2021
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
Commission Regulation (EC) No 735/2017; B.49
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Only healthy, non-smoking donors and not receiving medication were used. In this study, both
in the 1st and 2nd Experiment a 28-year old male donor was used. In the 3rd Experiment blood
was taken from a 30-year old male and in the 4th Experiment blood from a 33-year old female
donor was used.
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
The S9 fraction was prepared according to Ames et al. (7) at BASF SE in an AAALAC-approved
laboratory in accordance with the German Animal Welfare Act and the effective European
Council Directive.
At least 5 male Wistar rats [Crl:WI(Han)] (200 - 300 g; Charles River Laboratories Germany
GmbH, Sulzfeld, Germany) received 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone
orally (both supplied by Sigma-Aldrich, Taufkirchen, Germany) each on three consecutive
days.
During this time, the animals were housed in polycarbonate cages: central air conditioning with
a fixed range of temperature of 20 - 24°C and a fixed relative humidity of 45 - 65%. The
day/night rhythm was 12 hours: light from 6 am to 6 pm and dark from 6 pm to 6 am.
Standardized pelleted feed and drinking water from bottles were available ad libitum.
24 hours after the last administration, the rats were sacrificed, and the induced livers were
prepared using sterile solvents and glassware at a temperature of +4°C. The livers were
weighed and washed in a weight-equivalent volume of a 150 mM KCl solution (1 mL ≙ 1 g wet
liver), then homogenized in three volumes of KCl solution. After centrifugation of the
homogenate at 9000 x g for 10 minutes at +4°C, 5-mL portions of the supernatant (S9 fraction)
was stored at -70°C to -80°C.
The sterility of the S9 fraction was determined by incubating 1 mL S9 fraction on an agar plate
at 37°C for 48 hours. The content of protein was measured with an appropriate method (e.g.
Bradford). To check the activity of the S9 fraction a bacterial reverse mutation assay according
to Ames et al. (7) was performed.
Test concentrations with justification for top dose:
Following the requirements of the current OECD Guideline 487 a test substance with defined
composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, as initially the content of the test substance (approx. 20%) was assumed the max concentration was set to 10000 µg/ml for first and second experiment. However, within experiment 3 the exact content of the test substance (26.2%) was used (Top concentration 7575.8µg/ml).
Vehicle / solvent:
The test substance is an aqueous solution, therefore culture medium (DMEM/F12) was used
as the most suitable vehicle.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
colchicine
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Dublicates
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
The activated cell cultures were pooled and centrifugated in 10 mL aliquotes at 900 g for 5 minutes.
After centrifugation the supernatant (culture medium) was removed and the cells suspended in 9 mL test substance dilution in culture medium. A separate aliquote of the test substance dilution was incubated with the cultures to determine test substance precipitation at the end of exposure. At the end of the exposure period, the cells were transferred in tubes, centrifuged for 5 minutes at 900 g and resuspended in HBSS (Hanks Balanced Salt Solution). Washing of the cells was repeated at least once. Then the cells were centrifuged at (900 g, 5 min) and resuspended in
DMEM/F12 medium with 10% [v/v] FCS and transferred into 25 cm² cell culture flasks. After a recovery period of 16 h Cyt B (see 3.2.2) was added to the cultures and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% relative humidity for 20 hours. In case of continuous treatment of the cultures Cyt B was added directly after 20 h of treatment and further cultured as described above.


TREATMENT AND HARVEST SCHEDULE:
The cells were transferred into tubes, centrifugated at 900 g for 5 min and washed with HBSS. After washing, the cells were centrifuged (900 g, 5 min) and suspended in 0.0375 M KCl (37°C), incubation for 20 minutes at 37°C. After the hypotonic treatment, the cells were fixed by adding of fixative (19 parts methanol and 1 part acetic acid). The cells were centrifuged (900 g, 5 min, 4°C) and suspended in fresh fixative and incubated for 20 min at 4°C. The fixation step will be repeated twice. After the last fixation step, the cells can be centrifugated directly (900 g, 5 min, 4°C), suspended in 1-2 mL fresh fixative and spread on slides. The slides were dipped in deionized water, the cells were pipetted on the slide and fixed by passing through a flame.
The cells were stained with May-Grünwald (3 min) and 10% [v/v] Giemsa (in Titrisol, pH 7.2, 10 min) and mounted.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
Micronucleus analysis
The slides were scored microscopically. As a rule, at least 1000 binucleated cells per culture, in total at least 2000 binucleated cells per test group, were evaluated for the occurrence of micronuclei.
The analysis of micronuclei was carried out according to the following criteria of Countryman and Heddle.
- The diameter of the micronucleus was less than 1/3 of the main nucleus
- The micronucleus was not linked to the main nucleus and was located within the cytoplasm of the cell.
- Only binucleated cells were scored.
Slides were coded randomly before microscopic analysis with an appropriate computer program. Cultures with few isolated cells were analyzed for micronuclei.
Proliferation Index (CBPI)
The cytokinesis-block proliferation index (CBPI) is a direct measure of the proliferative activity of the cells and it was determined in 500 cells per culture (1000 cells per test group). This value indicates the average number of cell cycles per cell during the period of exposure to the actin polymerization inhibitor Cyt B.
The number of mononucleated, binucleated and multinucleated cells were recorded and the CBPI was calculated using the following formula: CBPI = ((No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells))/(Total number of cells)
The CBPI was used to calculate the % cytostasis (relative inhibition of cell growth compared to the respective vehicle control group) - a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytostasis.
% Cytostasis = 100 - 100 {(CBPIT - 1) / (CBPIC - 1)}
T = test substance treated culture C = vehicle control culture


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: Cytotoxicity Proliferation index cytostasis

Rationale for test conditions:
Following the requirements of the current OECD Guideline 487 a test substance with defined
composition should be tested up to a maximum concentration of 2 mg/mL, 2 μL/mL or 10 mM, taking into account the current guidelines and the initial information regarding the content of
the test substance (approx. 20%) a top concentration of 10000 mg/ml was choosen.
Evaluation criteria:
Assessment criteria
A test substance is considered to be clearly positive if all following criteria are met:
• A statistically significant increase in the number of micronucleated cells was obtained.
• A dose-related increase in the number of cells containing micronuclei was observed.
• The number of micronucleated cells exceeded both the concurrent vehicle control value and
the range of our laboratory’s historical negative control data (95% control limit).
A test substance is considered to be clearly negative if the following criteria are met:
• Neither a statistically significant nor dose-related increase in the number of cells containing
micronuclei was observed under any experimental condition.
• The number of micronucleated cells in all treated test groups was close to the concurrent
vehicle control value and within the range of our laboratory’s historical negative control data
(95% control limit).
Statistics:
An appropriate statistical analysis was performed. The proportion of cells containing
micronuclei was calculated for each test group. A comparison of the micronucleus rates of each
test group with the concurrent vehicle control group was carried out for the hypothesis of equal
proportions (i.e. one-sided Fisher's exact test, BASF SE).
If the results of this test were statistically significant compared with the respective vehicle
control (p ≤ 0.05), labels (s) were printed in the tables.
In addition, a statistical trend test (SAS procedure REG (16)) was performed to assess a
possible dose-related increase of micronucleated cells. The used model is one of the proposed
models of the International Workshop on Genotoxicity Test procedures Workgroup Report (17).
The dependent variable was the number of micronucleated cells and the independent variable
was the concentration. The trend was judged as statistically significant whenever the one-sided
p-value (probability value) was below 0.05.
However, both, biological and statistical significance were considered together.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
slight increase in cytotoxicity within concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
According to the results of the present in vitro micronucleus assay, the test substance Zincphthalocyanine
sulphonate in water did not lead to a biologically relevant increase in the
number of micronucleated cells either without S9 mix or after the addition of a metabolizing
system in four experiments performed independently of each other. In experiments 1 and 2 the
highest tested concentration (10000 μg/mL) corresponded (after receipt of the analytical data
and based on a content of 26.2%) to a test substance concentration of 2620 μg/mL. Therefore,
the positive trend analysis demonstrated in experiment one as well as the significantly increase
value (1.3%) in Experiment 2 were obtained at levels beyond the limit concentration given by
the OECD 487 guideline (2000 μg/mL). Furthermore, the positive trend analysis found in the
1st Experiment without S9 mix after pulse treatment could not be reproduced in the 4th
experiment. Additionally, the statistically and dose dependent increase found in the 2nd
Experiment without S9 mix after continuous treatement were also not reproducable in the 3rd
Experiment. Therefore, the findings in Experiments 1 and 2 in the absence of S9 mix are
considered as irrelevant.
The number of micronucleated cells in the vehicle control groups were nearby our historical
negative control data range (95% control limit) and, thus, fulfilled the acceptance criteria of this
study.
The proficiency of the laboratory to perform the micronucleus test in primary human
lymphocytes was demonstrated by the laboratory’s historical control database on vehicle and
positive controls and by X-bar chart to identify the variability of the vehicle control data.
The increase in the frequencies of micronuclei induced by the positive control substances
MMC, Colchicine and CPA clearly demonstrated the sensitivity of the test system and/or the
metabolic activity of the S9 mix employed. The values were compatible the range of the
historical positive control data and, thus, fulfilled the acceptance criteria of this study.

TREATMENT CONDITIONS


Osmolality and pH values were not relevantly influenced by test substance treatment.
In this study, in the absence and the presence of S9 mix, no precipitation in culture medium was observed microscopically or macroscopically up to the highest applied test substance concentration.


CYTOTOXICITY


After 4 hours treatment no relevantly reduced proliferative activity was observed in the absence of S9 mix in the 1st and 4th Experiment and in the presence of S9 mix in the 1st Experiment. After 20 hours continuous test substance treatment without S9 mix a dose dependent reduced cell proliferation was observed in the 2nd and 3rd Experiment. In Experiment 2 the CBPI cytostasis was 16.9, 17.7, 26.2 and 27.4% after treatment with 1714.7, 3086.4, 5555.6, 10000 μg test substance/ mL. In Experiment 3 the CBPI cytostasis was 14.0, 16.4, 21.6 and 31.3% after treatment with 1299.0, 2338.2, 4208.8 and 7575.8 μg test substance/ mL.


GENOTOXICITY - MICRONUCLEUS ANALYSIS

In the 1st Experiment in the absence of S9 mix the micronucleus rates of pulse treated cells
ranged between 0.6% and 0.9% micronucleated cells. The respective negative control value
was 0.3% micronucleated cells. All values were clearly within the range of the 95% control limit
of our historical negative control data range and not statistically significantly higher than the
respective vehicle control value (0.3% micronucleated cells). The trend analysis, however,
showed a concentration related increase in the mutation frequencies in the 1st Experiment. To
clarify these results a repeat experiment (4th Experiment) was perfomed to assess the
relevance of the data observed in experiment one. In the 4th Experiment micronucleus values
between 0.5% and 0.6% were obtained. The concurrent negative control was 0.4%. In this
experiment all evaluated values were within the range of the 95% control limit of the historical
negative control data (0.2 - 0.9% micronucleated cells; see Appendix 5), not significantly higher
than the respective vehicle control value and a dose dependency assessed by trend analysis
was not observed.
In the 2nd Experiment after continuous test substance treatment in the absence of metabolic
activation a dose dependent increased number of micronucleated cells was observed in groups
tested with 3086.4, 5555.6 and 10000.0 μg/mL (0.3%, 0.8 and 1.3% micronucleated cells,
respectively). The value at 10000 μg/mL was additionally statisitically increased compared to
the concurrent negative control value (0.4% micronucleated cells) and above the the range of
the 95% control limit of the historical negative control data (0.2 - 0.9% micronucleated cells;
see Appendix 5). To clarify these findings a repeat experiment (3rd Experiment) was perfomed.
In the 3rd Experiment micronucleus rates of 0.5%, 0.7% and 0.6% were observed at 2338.2, 4208.8 and 7575.8 μg/mL. The concurrent vehicle control was 0.4%. All values were within the
range of the 95% control limit of the historical negative control data (0.2 - 0.9% micronucleated
cells; see Appendix 5) and not significantly higher than the respective vehicle control value. A
dose dependency as obtained in the 2nd Experiment could not be confirmed.
The positive control substances MMC (without S9 mix 0.04 and 0.31 μg/mL), Colchicine
(without S9 mix: 0.05 μg/mL) and CPA (with S9 mix; 2.50 μg/mL) induced statistically
significantly increased micronucleus frequencies in all independently performed experiments.
In this study, in the absence and presence of metabolic activation the frequencies of
micronucleated cells (4 h without S9 mix: 6.6% and 10.6% micronucleated cells; 20 h without
S9 mix: 3.0% and 6.1% micronucleated cells for MMC and 2.2% and 3.6% for Colchicine in the
2nd and 3rd Experiment respectively); with S9 mix 3.5% micronucleated cells were compatible
to the historical positive control data range.

Conclusions:
Under the experimental conditions chosen here, the conclusion is drawn that Zincphthalocyanine
sulphonate in water has no potential to induce micronuclei (clastogenic and/or aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.
Executive summary:

The substance Zinc-phthalocyanine sulphonate in water was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Four independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Taking into account the current guidelines, the following concentrations were selected for the main experiments. Test groups printed in bold type were evaluated for the occurrence of
micronuclei:
1st Experiment
4 hours exposure, without S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
4 hours exposure, with S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
2nd Experiment
20 hours exposure, without S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
3rd Experiment
20 hours exposure, without S9 mix
0; 123.7; 222.7 ,400.9; 721.7; 1299.0; 2338.2; 4208.8; 7575.8 μg/mL
4th Experiment
4 hours exposure, without S9 mix
0; 123.7; 222.7 ,400.9; 721.7; 1299.0; 2338.2; 4208.8; 7575.8 μg/mL
The selected concentrations in experiment 1 and 2 were based on a test substance content of approx. 20%. However, after receipt of the analytical data, the content was shown to be 26.2%. Thus, all test substance concentrations in the repeat experiments (Experiments 3 and 4) were selected based on a purity of 26.2%. Thus, the highest tested concentration in Experiment 3 and 4 (7575.8 μg/mL) corresponds to approx. 2 mg test substance /mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells
for each test group.
In this study, culture medium was selected as vehicle. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei.
Dose-dependently increased cytotoxicity indicated by clearly reduced proliferation index (CBPI) was observed in cultures continuously treated in the absence of S9 mix in the 2nd and 3rd Experiment.


On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.


Thus, under the experimental conditions described, Zinc-phthalocyanine sulphonate in water is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Based on the available genotoxicity studies with ZPS and the knowledge on the genotoxicity profile of zinc, ZPS is not considered genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: gene mutation
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Canton-S wild type
Sex:
male
Route of administration:
other: feeding and injection
Vehicle:
water (feeding study); 0.7% aqueous NaCl (injection study)
Duration of treatment / exposure:
3 days (feeding)
single exposure (injection)
Frequency of treatment:
daily (feeding study)
once (injection)
Post exposure period:
1 day (injection)
Remarks:
Doses / Concentrations:
250 µL/mL (feeding)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.2 µL/mL (injection)
Basis:
other: actual received
No. of animals per sex per dose:
15 (feeding)
Control animals:
yes, concurrent vehicle
Positive control(s):
dimethylnitrosamine (DMN):
100 ppm (feeding)
500 ppm (injection)
Tissues and cell types examined:
A standard genetic scheme (Basc females x Canton-S wild type males) was employed and germ cells which were post-meiotic at the time of exposure were tested for lethal mutations.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Feeding: approximately 8% mortality was observed, no male sterility was induced;
Injection: approximately 9% mortality was observed, no male sterility was induced.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, near-guideline study, available as unpublished report, no restrictions, fully adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley Inc., Frederick, MD
- Age at study initiation: 9-10 weeks
- Weight at study initiation: 267-339 g (males), 206-259 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Diet: Rodent chow, ad libitum
- Water: from a municipal water supply, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12
Route of administration:
oral: gavage
Vehicle:
distilled water
Details on exposure:
Male and female Sprague-Dawley rats were treated with the substance at dose volumes of 1.6, 5.3 or 16 mL/kg body weight which was given as a single gavage administration. A cell cycle kinetics study indicated that the substance had no adverse effects on bone marrow cell cycle kinetics following acute administration of the high dose level.
Duration of treatment / exposure:
single administration
Frequency of treatment:
once
Post exposure period:
6 or 12 hours
Remarks:
Doses / Concentrations:
1.6, 5.3, 16 mL/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
other: yes, distilled water
Positive control(s):
cyclophosphamide (CP)
2'-5'-bromodeoxyuridine (BrdU)
Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
Bone marrow cells arrested in the metaphase and collected 6 or 12 hours after administration were examined microscopically for numerical and structural chromosome aberrations.
Evaluation criteria:
The test article is considered to induce a positive response when the number of aberrations per cell at any dose or doses is significantly increased relative to the vehicle centrol. A significant trend test (p≤0.05, Cochran-Armitage Test) only, with no single dose significantly increased, is considered equivocal.
Statistics:
Male and female animals ware analyzed separately. The Fisher's Exact Test was used to compare the percentage of aberrant ceIls between each animal and it's corresponding vehicle control group. The Wilcoxon's Rank Sum Test was used to compare the average number of aberrations per cell per animal in each treatment group to the appropriate vehicle control group. The Cochran-Armitage Trend Test was employed to test for evidence of a dose response between groups at each sacrifice time point.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The substance did not induce numerical and structural chromosome aberrations.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro data


ZPS was tested in a GLP compliant Ames reverse mutation assay according to OECD guideline 471 using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100 and Escherichia coli WP2 uvr A at 33 to 5000 µg/plate (two independent experiments (standard plate incorporation test and pre-incubation test) and each concentration was tested in triplicate) with and without metabolic activation (RCC Ltd., 2001). The plates incubated with ZPS showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No relevant toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with ZPS at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, under the experimental conditions reported, ZPS did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.


 


In a second GLP compliant reverse mutation assay according to OECD 471 using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and TA1538, ZPS was tested at concentrations of 0.2 to 20 µL/plate (Microbiological Associates, Inc., 1986). Under the conditions of this test, the substance did not cause a positive response on any of the tester strains with or without metabolic activation.


 


ZPS was tested in the Unscheduled DNA Synthesis Test (GLP compliant and performed according to a protocol similar to OECD guideline 482) using rat primary hepatocytes. The test article was tested at nine dose levels ranging from 0.003 µL/mL to 3.0 µL/mL (Microbiological Associates, Inc., 1987). Dose levels 0.01 to 0.6 µL/mL were fully evaluated, dose levels 1.0 - 3.0 µL/mL caused too much toxicity to evaluate the nuclei for UDS. The results of the UDS assay indicate that under the test conditions, the test article did not cause a significant increase in the mean number of net nuclear grain counts (i.e., an increase of at least 5 counts over the control), at any dose level. Therefore, the test article is considered negative in this study.


ZPS was also tested in a GLP-compliant mouse lymphoma L5178Y cells (TK assay tested according to OECD guideline 476) (Sitek Research Laboratories, 1987).Cultures were treated at concentrations of 1.0, 8.0, 15, 22, 29, 36, 43 and 50 µL/mL in the absence of exogenous activation and at concentrations of 0.02,0.16, 0.30,0.44,0.58, 0.72, 0.86 and 1.0 µL/mL in the presence of S-9 mix. The results indicated that in the presence and absence of exogenous activation, the test article induced a significant increase in the mutant frequency of treated cultures as compared to the mutant frequency of the corresponding solvent control cultures. The response was dose dependent.


Further Zinc-phthalocyanine sulphonate in water was assessed for its potential to induce micronuclei in primary human lymphocytes in vitro (clastogenic or aneugenic activity). Four independent experiments were carried out, with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation). Taking into account the current guidelines, the following concentrations were selected for the main experiments. Test groups printed in bold type were evaluated for the occurrence of
micronuclei:
1st Experiment
4 hours exposure, without S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
4 hours exposure, with S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
2nd Experiment
20 hours exposure, without S9 mix
0; 163.3; 294.0; 529.2; 952.6; 1714.7; 3086.4; 5555.6; 10000.0 μg/mL
3rd Experiment
20 hours exposure, without S9 mix
0; 123.7; 222.7 ,400.9; 721.7; 1299.0; 2338.2; 4208.8; 7575.8 μg/mL
4th Experiment
4 hours exposure, without S9 mix
0; 123.7; 222.7 ,400.9; 721.7; 1299.0; 2338.2; 4208.8; 7575.8 μg/mL
The selected concentrations in experiment 1 and 2 were based on a test substance content of approx. 20%. However, after receipt of the analytical data, the content was shown to be 26.2%. Thus, all test substance concentrations in the repeat experiments (Experiments 3 and 4) were selected based on a purity of 26.2%. Thus, the highest tested concentration in Experiment 3 and 4 (7575.8 μg/mL) corresponds to approx. 2 mg test substance /mL. A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells
for each test group.
In this study, culture medium was selected as vehicle. The negative controls gave frequencies of micronucleated cells within our historical negative control data range for primary human lymphocytes. The positive control substances, Mitomycin C (MMC), Colchicin and Cyclophosphamide (CPA), led to the expected increase in the number of cells containing micronuclei.
Dose-dependently increased cytotoxicity indicated by clearly reduced proliferation index (CBPI) was observed in cultures continuously treated in the absence of S9 mix in the 2nd and 3rd Experiment.


On the basis of the results of the present study, the test substance did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system.


Thus, under the experimental conditions described, Zinc-phthalocyanine sulphonate in water is considered to have no chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in primary human lymphocytes in the absence and the presence of metabolic activation.


 


 


 


In vivo data


A GLP compliant bone marrow chromosome aberration assay according to a protocol similar to OECD guideline 475 was performed with rats (Microbiological Associates, Inc., 1989). Male and female Sprague-Dawley rats were treated with the substance at dose volumes of 1.6, 5.3 or 16 mL/kg body weight which was given as a single gavage administration. All animals receiving 16 mL/kg and one male receiving 5.3 mL/kg exhibited dark blue-green faeces, approximately 5 hours after dosing for the 8 hour harvest. All animals receiving the substance exhibited dark blue-green faeces approximately 10 hours after dosing for the 12 hour harvest. All animals receiving distilled water appeared normal throughout the study. No significant reduction in the rate of body weight gain was observed among test article-treated animals.


There was no apparent change in ploidy in any of the treated animals. ZPS had no apparent effect on the mitotic index. The percentage of cells containing one or more aberrations was not significantly increased relative to the vehicle control in any of the test article treated animals. The mean aberrations per cell per animal were also not significantly increased regardless of dose or sacrifice time. The negative and positive controls fulfilled the requirements for determination of a valid test.


Under the conditions of this assay, ZPS, was negative in the acute cytogenetics assay using male and female Sprague-Dawley rats.


 


Asex-linked recessive lethal (SLRL) assay was performed in Drosophila melanogaster (University of Wisconsin, 1991). Adult Canton-S males were subjected to a 3 day feeding exposure to 250 µL/mL ZPS. Other Canton-S males were injected with ZPS at a concentration of 0.2 µL/mL. Exposure via feeding induced approximately 8% mortality and no male sterility was induced. Exposure via injection induced approximately 9% mortaility and also no male sterility was induced. The Canton-S males were mated to Basc females and germ cells which were post-mitotic at the time of exposure were tested for lethal mutations. ZPS did not induce mutations in the post-meiotic germ cells of Drosophila melanogaster males when administered by feeding or injection.


 


Conclusion


From the toxicokinetic studies with ZPS, it is known that zinc as measured by 65Zn is absorbed to a small extent after oral absorption and that the organic part of the ZPS molecule is most likely excreted without absorption. The very small amount of absorption as measured by 14C (organic part of the molecule) after oral and dermal absorption could be accounted for by absorption of radiochemical impurities in the dosing preparation.


Zinc is known to give (false) positive results in in vitro genetic toxicity studies. In the EU Risk Assessment Report of zinc (http://esis.jrc.ec.europa.eu/doc/existing-chemicals/risk_assessment/DRAFT/R072_0609_env_hh.pdf), a negative (Amacher and Paillet, 1980) and two positive results (Cameron, 1991; Adams and Kirkpatrick, 1994) were reported in mouse lymphoma assays.


However, the EU Risk Assessment Report concluded that there is no evidence for genotoxicity in vivo for zinc. Furthermore, zinc is an essential trace element.


Therefore, the positive result in the mouse lymphoma assay for ZPS is not considered relevant for the in vivo situation. Overall, based on the available other in vitro and in vivo genotoxicity studies with ZPS which all gave negative results and the knowledge on the genotoxicity profile of zinc, ZPS is not considered genotoxic.

Short description of key information:
Based on the available genotoxicity studies with ZPS and the knowledge on the genotoxicity profile of zinc, ZPS is not considered genotoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the results of the in vitro and in vivo tests, ZPS does not need to be classified according to Directive 67/548/EEC and according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.