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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

No signs of genotoxic effects in-vitro were observed, neither in bacterial systems nor in mammalian cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): Heliogen Blau K 6915
- Test substance No.: 00/0503-1
- Analytical purity: >= 98 g / 100 g
- Lot/batch No.: 00-0503-1
- Storage condition of test material: Room temperature
Target gene:
Salmonella typhimurium: Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).
Escherichia coli: Determination of the rate of induced back mutations of bacterial mutant from trypthophan auxotrophy (trp-) to tryptophan prototrophy (trp+).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the liver of male Sprague-Dawley rats (treated i.p. with 500 mg/kg bw Aroclor 1254 5 days before sacrifice), mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH7.4).
Test concentrations with justification for top dose:
1st experiment:
Standard plate test with and without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate
2nd experiment:
Preincubation test with and without S9-mix (S. typhimurium T1535 and TA100): 0, 20, 100, 500, 2500 and 5000 µg per plate
3rd experiment:
Preincubation test with and without S9-mix (S. typhimurium TA1535, TA98 and E. coli WP2 uvrA): 0, 20, 100, 500, 2500 and 5000 µg per plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO had been demonstrated to be a suitable vehicle in bacterial reverse mutation tests; historical control data are available for DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: With S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:
A standard plate test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure of the standard plate test (plate incorporation method) is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975) and Maron and Ames (Mut. Res. 113: 173-215, 1983)
Salmonella typhimurium:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42-45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.
Composition of the minimal glucose agar:
980 mL purified water
20 mL Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose, monohydrate.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) are counted.

Escherichia coli:
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation).
After mixing, the samples are poured onto minimal agar plates within approx. 30 seconds.
The composition of the minimal agar (SA1 selective agar) is based on the description of Green, M.H.L. and Muriel, W.J. (Mut. Res. 38: 3-32, 1976), with the exception of solution E (tryptophan solution), which has previously been added to the soft agar:
300 mL solution B (agar)
100 mL solution A (saline solution)
8 mL solution C (glucose solution)
10 mL solution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

PREINCUBATION TEST:
The experimental procedure is based on the method described by Yahagi et al. (Mut. Res. 48: 121-130, 1977) and Matsushima et al. (Factors modulating mutagenicity in microbial tests. In: Norpoth, K.H. and R.C. Garner, Short-Term Test Systems for Detecting Carcinogens. Springer Verlag Berlin, Heidelberg, New York, 1980):
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar is added and, after mixing, the samples are poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.

CONTROLS:
Negative control:
Parallel with each experiment with and without S9-mix, negative controls (solvent control, sterility control) were carried out for each tester strain in order to determine the spontaneous mutation rate.
Positive controls:
The following positive control substances were used to check the mutability of the bacteria and the activity of the S9-mix. With S9 mix: 2-aminoanthracene, 2-AA (2.5 µg/plate for all 4 Salmonella strains, 60 µg/plate for E. coli strain); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA 1535 and TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98), 4-nitroquinoline-1-oxide, 9-Aminoacridine, AAC (100 µg/plate for TA 1537), 4-NQO (5 µg/plate for E. coli WP2 uvrA).

TITER DETERMINATION:
The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.
In the standard plate test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. Test tubes containing 2 mL portions of soft agar containing maximal amino acid solution (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45°C, and the remaining components are added in the following order:
0.1 mL vehicle (without and with test substance)
0.1 mL fresh bacterial culture (dilution: 10E-6)
0.5 mL S9 mix
In the preincubation test, 0.1 mL of the overnight cultures is diluted to 10E-6 in each case. 0.1 mL vehicle (with and without test substance), 0.1 mL bacterial suspension and 0.5 mL S9 mix are incubated at 37°C for about 20 minutes using a shaker. Subsequently, 2 mL of soft agar containing maximal amino acid solution for titer determination (5 mM tryptophan or 5 mM histidine + 0.5 mM biotin) is added.
After mixing, the samples are poured onto the agar plates within approx. 30 seconds. After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies are counted.
Evaluation criteria:
Assessment criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if the number of revertants for all tester strains are within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
- The number of revertant colonies in the negative controls is within the range of the historical negative control data for each tester strain.
- The sterility control reveales no indication of bacterial contamination.
- The positive control substances both with and without S9 mix induce a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
- The titer of viable bacteria is >= 1x10exp+8/ml.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
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
Additional information on results:
TOXICITY:
No bacteriotoxic effect was observed.

SOLUBILITY:
Test substance precipitation was found from about 100 µg/plate onward.

MUTAGENICITY:
An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S-9 mix or after the addition of a metabolizing system in any bacterial strain tested.

Table 1: Maximum revertants/plate and corresponding test concentrations in thestandard plate test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

21 ± 2

23 ± 2

Test substance

23 ± 4 [2500]

25 ± 2 [100]

Positive Control

639 ± 15 [5; MNNG]

136 ± 33 [2.5; 2-AA]

S. typhimurium TA100

DMSO

108 ± 6 

109 ± 3

Test substance

104 ± 4 [20]

112 ± 5 [500]

Positive Control

887 ± 73 [5; MNNG]

809 ± 59 [2.5; 2-AA]

S. typhimurium TA98

DMSO

33 ± 2 

44 ± 4

Test substance

34 ± 5 [100]

57 ± 8 [5000]

Positive Control

712 ± 33 [10; NOPD]

572 ± 46 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

10 ± 2

10 ± 3

Test substance

9 ± 3 [20]

12 ± 4 [2500]

Positive Control

609 ± 100 [100; AAC]

95 ± 3 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

48 ± 7

45 ± 3

Test substance

45 ± 13 [20]

52 ± 11 [500]

Positive Control

734 ± 46 [5; 4-NQO]

228 ± 26 [60; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

 

Table 2: Maximum revertants/plate and corresponding test concentrations in thepreincubation test:

    

Strain

Tested compound

Maximum revertants/plate [corresponding dose unit in µg/plate]

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

17 ± 2

17 ± 1

Test substance

17 ± 2 [100]

16 ± 2 [20]

Positive Control

795 ± 40 [5; MNNG]

203 ± 6 [2.5; 2-AA]

S. typhimurium TA100

DMSO

106 ± 6

109 ± 7

Test substance

108 ± 6 [20]

107 ± 4 [20]

Positive Control

1094 ± 105 [5; MNNG]

889 ± 68 [2.5; 2-AA]

S. typhimurium TA98

DMSO

28 ± 5

31 ± 3

Test substance

30 ± 7 [20]

41 ± 4 [5000]

Positive Control

877 ± 68 [10; NOPD]

632 ± 63 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

9 ± 2

9 ± 2

Test substance

9 ± 3 [100]

9 ± 1 [20]

Positive Control

436 ± 18 [100; AAC]

94 ± 5 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

38 ± 5

29 ± 5

Test substance

39 ± 4 [5000]

35 ± 6 [2500]

Positive Control

483 ± 102 [5; 4-NQO]

223 ± 7 [60; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

Conclusions:
According to the results of the present study, the test substance did not lead to an increase in the number of his revertant colonies either with or without S9-mix in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Batch no. 21993
- Storage conditions: Room temperature
- Name: Fastogen Blue 10GN
- Fine violet powder
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Details on mammalian cell type (if applicable):
obtained from the JCRB Cell Bank.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 from Aroclor1254-induced rats
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 μg/ml (preliminary test)
1250 - 5000 μg/ml (main test)
Vehicle / solvent:
Cell culture medium

The test material was found to be poorly soluble in dimethyl sulphoxide and acetone, but to form a doseable suspension in culture medium at concentrations of 50 mg/ml and below. Accordingly, suspensions ofthe test material for use in testing were prepared in culture medium immediately before addition to test cultures. The suspensions of maximum concentration was prepared initially and lower concentrations were prepared by dilution in culture medium. All concentrations cited in this report are stated in terms of the material as supplied
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (suspension)

DURATION
- Preincubation period: 24h
- Exposure duration: without S-9 mix cells were exposed continuously for 6, 24 or 48 hours, with S-9 mix exposure was limited to 6 hours; cells exposed for 6 hours were cultured in fresh medium for a further 18 hours before harvesting.
- Expression time (cells in growth medium): 18h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 or 48h


SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 1.5

NUMBER OF CELLS EVALUATED: 1000

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS: A sample ofthe laboratory stock culture was tested in December 1996 and was found to be mycoplasma free.
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Rationale for test conditions:
The test material was found to be poorly soluble in dimethyl sulphoxide and acetone, but to form a doseable suspension in culture medium at concentrations of
50 mg/ml and below. Accordingly, suspensions ofthe test material for use in testing were prepared in culture medium immediately before addition to test
cultures. The suspensions of maximum concentration was prepared initially and lower concentrations were prepared by dilution in culture medium. All
concentrations cited in this report are stated in terms of the material as supplied; no correction has been made for purity or activity below 100%.
Evaluation criteria:
The test material is considered to be clastogenic in this test if the following conditions are
met:
- statistically significant increases in the frequency of metaphases with aberrant
chromosomes (excluding gap-type aberrations) are observed at one or more test
concentrations
- the increases exceed the historical negative control range at this laboratory
- the increases are reproducible between replicate cultures and between tests
- the increases are not associated with !arge changes in pH or osmolarity of the treatment
medium or extreme toxicity (which can cause non-specific effects)
- evidence of a dose-response relationship, or increases at both sampling times will be
considered to support the conclusion.
The biological significance of gap-type aberrations is questionable and increases observed
only when gaps are included in the analysis will not be considered to be conclusive
evidence of clastogenic activity.
Statistics:
The Fisher Exact Probability test is a useful technique for analysing data when comparing
two independent samples. lt is used when the observed events all fall into one or other of
two mutually exclusive classes. The test determines whether the two groups differ in the
proportions with which they fall into the two classifications.
The frequency of aberrant metaphases for each treatment group was compared with the
corresponding solvent control group value using a one-tailed test.
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: substance is insoluble
- Precipitation: substance is applied as a suspension
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: yes, substance is not cytotoxic

Preliminarv toxicity test - Mitotic indices: Presence of S-9 mix - 6 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
Culture medium 1 1005 76 7.6 5.8
(-) 2 1002 39 3.9
Test material 3 1028 40 3.9 4.1 29
(8) 4 1047 45 4.3
Test material 5 1018 52 5.1 5.4 7
(40) 6 1033 59 5.7
Test material 7 1018 48 4.7 3.6 38
(200) 8 1032 25 2.4
Test material 9 1011 37 3.7 4.9 16
(1000) 10 1028 63 6.1
Test material 11 1040 85 8.2 6.8 Increase
(5000) 12 1027 55 5.4
Mitotic index = number of metaphases x 100

Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 6 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
Culture medium 13 1014 305 30.1 25.5
(-) 14 1011 210 20.8
Test material 15 1005 220 21.9 19.3 24
(8) 16 1021 170 16.7
Test material 17 1001 480 48.0 35.9 increase
(40) 18 1002 238 23.8
Test material 0.19 1016 376 37.0 23.8 7
(200) 20 1005 107 10.6
Test material 21 1030 302 29.3 26.7 increase
(1000) 22 1019 245 24.0
Test material 23 1034 130 12.6 16.4 36
(5000) 24 1058 214 20.2

Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 24 hour exposure. 24 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
#
Culture medium 25 1009 432 42.8 42.5 -
(-) 26 1028 434 42.2
Test material 27 1016 321 31.6 34.7 18
(8) 28 1020 385 37.7
Test material 29 1.010 344 34.1 35.8 16
(40) 30 1013 380 37.5
Test material 31 1018 443 43.5 39.1 8
(200) 32 1013 351 34.6
Test material 33 1014 416 41.0 35.2 17
(1000) 34 1021 299 29.3
Test material 35 1009 222 22.0 23.9 44
(5000) 36 1007 260 25.8

 Preliminary toxicity test - Mitotic indices: Absence of S-9 mix - 48 hour exposure. 48 hour sampling time
Treatment (µg/ml) Culture number Number of cells Number of metaphases Mitotic index* Mean mitotic index %
Reduction
#
Culture medium 37 1026 64 6.2 5.5                -
(-) 38 1045 49 4.7
Test material 39 1019 48 4.7 4.1 25
(8) 40 1037 35 3.4
Test material 41 1029 81 7.9 7.8 increase
(40) 42 1021 78 7.6
Test material 43 1018 68 6.7 5.9 increase
(200) 44 1008 50 5.0
Test material 45 1018 43 4.2 4.6 16
(1000) 46 1019 50 4.9
Test material 47 1007 66 6.6 7 increase
(5000) 48 1085 80 7.4
Conclusions:
non clastogenic in vitro
Executive summary:

The effects on chromosomal structure of exposure to the substance were investigated in cultured CHL (Chinese hamster lung) cells. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix):

without S-9 mix cells were exposed continuously for 6, 24 or 48 hours, with S-9 mix exposure was limited to 6 hours; cells exposed for 6 hours were cultured in fresh medium for a further 18 hours before harvesting.

Treatments were established by the addition oftest suspensions (in culture medium) to 24-hour cultures. Cell division was arrested by the addition ofthe spindle poison, Colcemid, two hours before the cells were harvested; slides were then prepared for microscopic analysis.

All slides were scored for chromosomal aberrations. The highest concentration scored for chromosomal aberrations (5000 μg/ml) is the highest required by EC, OECD, US and Japanese regulatory test guidelines.

One hundred metaphases were analysed from all selected cultures. Treatment with the test substance did not produce biologically or statistically significant increases in the frequency of metaphases with aberrant chromosomes at any concentration tested,

compared to vehicle control values (p>0.05 both including and excluding gap-type aberrations), at any sampling time, either in the presence or absence of S-9 mix.

The known clastogens, Mitomycin C and cyclophosphamide, induced significant increases in the frequency of metaphases with aberrant chromosomes, compared to the vehicle control values, at both sampling times in both cytogenetic tests (p<0.001 in all cases), thus demonstrating the sensitivity ofthe test procedure, and the metabolic activity of the S-9 mix employed.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells in vitro
Specific details on test material used for the study:
- Substance type: pigment
- Purity: 95%
- Physical state: solid
- Lot/batch No.: 841094
- Expiration date of the lot/batch: not indicated. PB 15 does not degrade under storage conditions.
- Stability under test conditions: yes
- Storage condition of test material: room temperature
Target gene:
none
Species / strain / cell type:
primary culture, other: rat hepatocytes, male animal
Metabolic activation:
not applicable
Metabolic activation system:
Primary hepatocytes do not need an additional metabolic activation system.
Test concentrations with justification for top dose:
60,0, 12,0, 2,40 and 0,48 ug/ml

DNA-repair test (repeat experiment):
0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 20, 40 and 60 ug/ml

The top dose is determined by precipitation.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
Remarks:
50 and 25 mM
Details on test system and experimental conditions:
Origin of primary hepatocytes:
Species/Sex: Rat/male
Strain: Tif: RAIf(SPF)
Origin: Tierfarm, Sisseln, Switzerland
Age/Body weight: adult/170 - 350 gr
Temperature : 21 + 2°C
Rel. humidity: 60 ± 10%
Lighting : 12 hours daily

Primary hepatocytes are isolated from adult male rats (Tif.RAIf (SPF), weight 170-350 g) induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A. by in situ-collagenase perfusion according to the method described by M.N. Berry and D.S. Friend, 1969 (Ref.5) as modified by L.R. Schwarz et al, 1979. The procedure is as follows: the liver is perfused in situ through the portal vein for about eight minutes with the following medium: 121 mM NaCI, 6 mM KCI, 0.6 mM MgSO4, 12 mM NaHCO3, 0.74 mM KH2PO4, 5 mM glucose. The medium is aerated with carbogen (95% O2, 5% CO2), it temperature is 37°C, the pH about 7.4. After insertion of a canule into the thoracal part of the vena cava, the perfusion is continued for further 15-20 minutes by recirculation of the medixim, which is supplemented with 0.05% collagenase and 2.5 mM CaCl2. The liver is then carefully excised and placed into a dish containing calcium-free Hanks' solution (4°C). After opening the Glisson's capsule, the cells are dispersed by gently shaking
of the liver in the solution. The cells are then filtered (mesh width of 61 vim) and washed twice with calcium-free Hanks'
solution (sedimentation rate of 50 g for 3 minutes at 2°C). Finally, the cells are suspended in Williams' medium E and analysed
for viability by trypan-blue exclusion. The viability of hepatocytes prepared in this way is generally greater than 90%. The weight
of the rats sacrificed for the toxicity test and for the DNA-repair tests amounted to 285 g, 190 g and 218 g, respectively.

Freshly isolated male rat hepatocytes are cultured in WILLIAMS' Medium E containing 10% foetal bovine serum, 100 U/ml penicillin, 100 ug/ml streptomycin and 2.5 ug/ml amphotericin (culture medium) and incubated in a humidified atmosphere with 5 % CO2 at 37°C. A series of compartments in Multiplates containing gelatinized THERMANOX cover-slips are seeded with 4 x 10 exp5 cells per compartment (density 10 exp5 cells/ml; 4 ml/compartment). The cells are allowed to attach to the cover-slips during an attachment period of 1.5-2 hours. Unattached cells are then removed by washing with BSS. The cultures are then refed with culture medium (2 ml/compartment) and cultivated overnight (adhesion period).

Exposure period: 5h (concommittant with 3H-Thymidine)
The nuclei are swollen by treatment with 1% sodium citrate for ten min. Cells are fixed with ethanol/acetic acid, 3/1, v/v. The coverslips are mounted on microscope slides and prepared for autoradiography. The exposure time is 6 days. The autoradiographs are stained with hematoxylin-eosine.
Evaluation criteria:
Cells which were in the DNA-synthesis phase showed more than 120 silver grains/nucleus. The percentage of such cells were about 0.07 and 0.08 (Tables 2 and 4). These cells were excluded from the determination of the silver grain/nucleus count.

The test substance is generally considered to be active in the
DNA-repair test if one of the following conditions is met;
- The mean number of silver grains per nucleus in relation to the vehicle control is more than doubled at any concentration.
- The mean number of silver grains per nucleus in relation to the vehicle control shows a concentration dependent increase and at
least at one concentration a statistically significant increase in comparison with the vehicle control is demonstrated.
- The percental distribution range of silver grains per nucleus is shifted towards higher values if compared with the range of the vehicle control.

Assay acceptance criteria:
- The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
- The viability of the hepatocytes collected from the perfusion process should exceed 70 %.
- The labelling in the vehicle control cultures should not exceed an average of ten total grains/nucleus, or the number of the
vehicle control nuclei with more than eight silver grains should not exceed 10 %.
- The positive control should fulfil all the criteria given for a positive response.
- Grain count data for a given treatment must be obtained from at least two replicate cultures and at least 50 cells per culture.
- A minimum of four concentrations of the test substance, a negative and a positive control should be analysed for nuclear grain counts.
Statistics:
The values of silver grains per nucleus are compared using Duncan's multiple range test. Statistical significance is judged to be achieved
if the probability is less than 0.05.
Species / strain:
primary culture, other: rat hepatocytes
Remarks:
male animal
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Background determination
Treatment group concentration Silver grain per nucleus-equivalent area
control (medium) 0.40
control (Vehicle) 0.40
positive control DMN 100 mM 0.33
Test substance  60.0 µg/ml 0.53
Test substance 12.0 µg/ml 0.40
Test substance 2.4 µg/ml 0.13
Test substance 0.48 µg/ml 0.13

Treatment group concentration Silver grain per nucleus-equivalent area ± SD
control (medium) n.a. 1,47 ± 1,16
control (Vehicle) n.a. 1.29 ± 1.08
positive control DMN 100 mM 20.1 + 6.89
Test substance  60.0 µg/ml 2.06 ± 1,53
Test substance 12.0 µg/ml 1.87 + 1.11
Test substance 2.4 µg/ml 1.76 ± 1.36
Test substance 0.48 µg/ml 2.27 ± 1.37
Conclusions:
negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No signs of genotoxic effects in-vivo were seen, neither in the Micronucleus Test, nor in the Nucleus Anomaly Test or the Mouse Spot test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
yes
Remarks:
No plasma analytics. Tested doses exceeded limit dose, 1000 cells per animal scored (but acceptable, since two groups above limit dose scored)
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 13 143 (crude copper phthalocyanine)
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375
Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Ciba-Geigy Tierfarm, Sisseln, Switzerland
- Age at study initiation: females 6 to 10 weeks, males 4 to 9 weeks
- Weight at study initiation: females 21 to 32 g, males 22 to 33 g in tolerability test; females 20 to 27 g, males 20 to 26 g in mutagenicity test
- Diet: NAFAG No. 924
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 40 - 46 %
- Housing in air conditioned rooms
- Photoperiod: 12hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
0.5 % Carboxymethylcellulose (CMC), Hercules Comp., USA
Details on exposure:
Tolerability test:
A preliminary test was conducted to determine the highest dosage of the test material to be applied in the mutgenicity test (Doses / Concentrations:
200, 1000 and 5000 mg/kg bw). Three groups of 4 chinese hamsters were treated with 3 different single doses. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. The highest dose survived by all animals was used in the second part of the tolerability test.
In the second part, the animals were treated according to the scheme used in the mutagenicity test with consecutive doses. The observation period corresponded to the interval between administration and sacrifice of the animals in the mutagenicity test, plus one day. Depending on the outcome the highest dose causing no deaths was used as the highest in the mutagenicity test.

Mutagenicity test:
The test material was administered orally to groups of 6 female and 6 male animals each. Treatment consisted of daily one application on 2 consecutive days. 24 h after the second application the animals were sacrificed.
Duration of treatment / exposure:
48 h
Frequency of treatment:
two treatments on 2 consecutive days
Post exposure period:
24 h after the second application
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Tolerability test: 2 animals per sex per dose
Mutagenicity test: 6 animals per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (ENDOXAN): 128 mg/kg bw in 20 ml/kg bw 0.5 % CMC
Details of tissue and slide preparation:
Bone marrow was harvested from the shafts of both femurs and homogenized. Small drops were transferred on the end of a slide and spread out. 3 h later, the slides were stained in undiluted May-Grünwald solution/water for 2 min and in Giemsa´s 40 % for 20 min. After being rinsed in methanol 55 % for 5-8 sec and washed with water, the slides were cleaned in xylene and mounted in Eukitt.
The slides of three female and three male animals each of the negative and positive control group and of the groups treated with various doses of the test material were examined. 1000 bone marrow cells each were scored per animal and the following anomalies were registered: single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in leucopoietic cells and polyploid cells.
Statistics:
The significance of difference was assessed by x2-test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In all dose groups the percentage of cells displaying anomalies of nuclei did not differ significantly from the negative control (0.1 %).
By contrast, the positive control yielded in a marked increase of the percentage of cells with anomalies (9.48 %).

Table 1: Percent of cells with anomalies of nuclei

 

Animal No.

Sex (m/f)

Single Jolly Bodies

Fragments of nuclei in erythrocytes

Micronuclei in erythrocytes

Micronuclei in leucopoietic cells

Polyploid cells

Total

negative control

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

0.2

 

 

 

 

0.2

4

m

0.1

 

 

 

 

0.1

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

cyclophosphamide

1

f

11.6

2.3

2.0

 

 

15.9

2

f

4.5

1.0

1.6

0.1

 

7.2

3

f

9.1

2.2

1.3

0.1

 

12.7

4

m

6.7

0.7

1.0

0.3

0.3

9.0

5

m

4.0

1.2

0.8

 

 

6.0

6

m

4.4

0.6

0.9

0.2

 

6.1

1250 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

 

 

 

 

 

0.0

3

f

0.1

 

 

 

 

0.1

4

m

 

 

 

 

 

0.0

5

m

0.1

 

 

 

 

0.1

6

m

0.1

 

 

 

 

0.1

2500 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

0.1

 

 

 

 

0.1

4

m

0.3

 

 

 

 

0.3

5

m

0.1

 

 

 

 

0.1

6

m

 

 

 

 

 

0.0

5000 mg/kg bw

1

f

 

 

 

 

 

0.0

2

f

0.2

 

 

 

 

0.2

3

f

 

 

 

 

 

0.0

4

m

0.1

 

 

 

 

0.1

5

m

 

 

 

 

 

0.0

6

m

0.1

 

 

 

 

0.1

Endpoint:
in vivo mammalian germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 484 (Genetic Toxicology: Mouse Spot Test)
Version / remarks:
adopted 23 Oct 1986
Deviations:
yes
Remarks:
Historical control data not included in the report. MDS not scored, Limit dose exceeded by factor of 5
GLP compliance:
yes
Type of assay:
mouse spot test
Specific details on test material used for the study:
- Name of test material (as cited in study report): TK 13 143 (crude copper phthalocyanine)
- Analytical purity: commercial grade
- Lot/batch No.: F 53 / H 91375
Species:
mouse
Strain:
other: C57/Bl/6, males: T-stock
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Bomholtgard Ltd. Denmark
- Age at study initiation: 3 - 4 months
- Weight at study initiation: females 20 - 23 g in toleerability test and 19 - 30 g in mutagenicity test; male body weight was not determined. (Males were not treated; males were included to mate with females and then only pregnant females were treated.)
- Assigned to test groups randomly: yes
- Diet: NAFAG No. 890 pellets standard diet, ad libitum
- Water: tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 48 - 56 %
- Air conditioned room
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
intraperitoneal
Vehicle:
Sesame oil was used as vehicle for the test material, Hank´s BSS was used as vehicle control for the positive control.
Details on exposure:
Tolerability test:
A preliminary test was conducted to determine the highest dosage of the test material to be applied in the mutgenicity test. 3 groups of 4 female mice were treated with 3 different single doses.The observation period lased 2 weeks. Depending on the outcome, the highest dose causing no deaths was used as the highest in the mutagenicity test or, if neccessary, the test was repeated with lower doses. Doses tested for tolerability were 200, 1000 and 5000 mg/kg bw.

Mutagenicity test:
One untreated male was placed in a cage with 2 untreated females. The females were inspected daily for successful mating. The day on which a vaginal plug was observed was designated as "day 1/2 of gestation". The females presumed to be pregnant were removed and the procedure was repeated for 4 consecutive days (4 mating nights). Subsequently the presumably pregnant females were uniformely distributed among the respective groups by random.
The test material preparation was administered intraperitoneally to groups of 71 successfully mated females. All presumably pregnant females were treated on the 10th day after conception. Treatment consisted of a single i.p. injection of the respective dose. The animals of the control group received vehicle only.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
until the birth of the offspring
Dose / conc.:
1 250 mg/kg bw/day (actual dose received)
Dose / conc.:
2 500 mg/kg bw/day (actual dose received)
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Tolerability test: 4 females per group
Mutagenicity test: 48 males and 96 females per group
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-ethylurea (ENU), 50 mg/kg bw in Hank´s BSS was administered intraperitoneally in parallel.
Tissues and cell types examined:
Animals treated as embryos were allowed to come to birth. The number of live and dead offspring was listed. The pups were inspected for external visible morphological changes. The examination upon spots began at the age of 12 - 14 days and was carried out twice per week during 3 weeks. 2 classes fo spots were distinguished and registered: pigmented and white spots, randomly distributed on the coat (recessive spots RS) and white mid-ventral spots (WMVS) within 5 mm of the mid-ventral line presumably arising from cell killing and thus not a result of mutagenic effects. Yellow, agouti-like spots in the vicinity of the mammae, genitalia, throat, axillary and inguinal areas and on the mid-forehead, which are presumed to result from misdifferentiation (MDS) are omitted from scoring. The pelts from the animals with spots were preserved.
Statistics:
The statisitcal analysis was conducted in 2 parts, Firstly the numbers of recessive spots in the control group and in the treatment groups were compared by a chi-squared test. Secondly, a test was carried out to determine whether the frequency of recessive spots increases with increasing doses.
If the effect of the substance increased with the dose, the trend test was preferable. The tests were applied on the condition that the proportions of recessive spots were constant over litters.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Tolerability test:
The dose of 5000 mg/kg bw was found to be the highest applicable in the mutagenicity test and was administered, together with 2 further doses, diminishing by a factor of 0.5.

Mutagenicity test:
From 71 presumably pregnant females per dose group, the following numbers actually were pregnant and gave birth to litters: Control: 56; 1250 mg/kg bw: 45, 2; 2500 mg/kg bw: 48; 5000 mg/kg bw: 44.
The average littersizes registered were: Control: 6.45; 1250 mg/kg bw: 5.93; 2500 mg/kg bw: 5.29; 5000 mg/kg bw: 6.07. 343 animals from the control group, 216, 171 and 169 animals from the groups, treated with 1250, 2500 and 5000 mg/kg bw were examined for colour spots.
The following percentages of animals with recessive (RS) and mid-ventral (WMVS) spots were recorded from gross observations:
RS: Control 0.29 %, 1250 mg/kg bw: 0.93 %, 2500 mg/kg bw: 0 %, 5000 mg/kg bw: 0 %
WMVS: Control 1.17 %, 1250 mg/kg bw: 3.24 %, 2500 mg/kg bw: 2.34 %, 5000 mg/kg bw: 1.78 %
In the positive control, the mean percentage of RS spots was 4.75 and of WMVS spots 2.71.
Statistical analysis for RS revealed the following results: Overall test X2 (3) = 3.82,p = 0.2819; Trend test (one-sided): Z = -0.7637, p = 0.7775

Table 1: THE EFFECT ON OFFSPRING FROM C57 Bl/6 x T CROSS TREATED IN UTERO

dose vehicle route of application treated females with vaginal plug % females with litters average litter size number of offspring examined offspring with recessive spots (total) % number of offspring with intraventral spots (total) %
negative control Sesame oil i .p . 71 62.0 6.1 169 0.0 0.0 3 1.8
1250 Sesame oil i .p . 71 63.4 5.9 216 2.0 0.9 7 3.2
2500 Sesame oil i .p . 71 67.6 5.3 171 0.0 0.0 4 2.3
5000 Sesame oil i .p . 71 62.0 6.1 169 0.0 0.0 3 1.8
50 mg/kg positive control Hank's i .p . 71 66.2 6.5 295 14.0 4.8 8 2.7

Table 2: Results of positive control experiment

dose (mg/kg bw) vehicle or N-Nitroso-N-ethylurea vehicle route of application treated females with vaginal plug % females with litters average litter size number of offspring examined offspring with recessive spots (total) % number of offspring with intraventral spots (total) %
negative control Hank's BSS i .p . 66 68.2 5.6 277 0.0 0.0 3 1.1
25 Hank's BSS i .p . 66 74.2 7.4 350 14.0 4.0 10 2.9
50 Hank's BSS i .p . 66 66.7 7.2 298 20.0 6.7 5 1.7
75 Hank's BSS i .p . 66 71.2 7.2 270 27.0 10.0 18 6.7
Conclusions:
Mated female mice received a single intraperitoneal injection of 1250, 2500 or 5000 mg/kg bw on day 10 of pregnancy. An similar number of litters and litter size was observed for all treatment groups indicating absence of embryotoxicity. The number of offspring with recessive spots (indicators of mutagenicity) was increased in the positive control group, but not in the treatment groups. The number of intraventral spots (indicators of toxicity) showed a higher a variability without dose-dependency.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The substance was non mutagenic in a GLP-compliant Ames test performed according to OECD guideline 471 (Dainippon 1994).

It was non clastogenic in a GLP-compliant in-vitro chromosome abberration test performed according to OECD guideline 473 (SunChemical 1997).

The substance is assessed to be non genotoxic in mammalian cells in vitro based on read-across. The read-across justifcation is attached to the endpoint study record.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity was observed in in vitro or in vivo studies. As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.