Copper chlorophthalocyanine

Endpoint summary

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.

CAS No. 147-14-8:

Gene mutation in bacteria

- S. typhimurium TA1535, TA1537, TA98 and TA100, with and without metabolic activation (Ames test): negative (comp. OECD 471; BASF AG 1985)

- S. typhimurium TA97, TA98, TA98, TA100 and TA102, with and without metabolic activation (Ames test): negative (comp. OECD 371, JETOC 1995)

- S. typhimurium TA98 and TA100, with and without metabolic activation (Ames test): positive presumably due to impurities (Hayatsu 1983, Val. 4)

Gene mutation in mammalian cells Mouse L5178Y cells with and without metabolic activation (Mouse lymphoma assay): negative (Manandhar 1982, Val. 4)

Cytogenicity in mammalian cells CHL cells (in vitro chromosomal aberration test), with and without metabolic activation: negative (acc. Guidelines for Screening Mutagenicity Testing of Chemicals, JETOC 1995).

Genome mutation in mammalian cells C3H/10T1/2 cells (cell transformation test): negative (Manandhar 1982, Val. 4) DNA damage and/or repair Rat hepatocytes (UDS assay): negative (Manandhar 1982, Val. 4)

CAS No. 1328-53-6:

Gene mutation in bacteria

- S. typhimurium TA1535, TA1537, TA98 and TA100 and E. coli WP2 uvrA, with and without metabolic activation (Ames test): negative (acc. Guidelines for Screening Mutagenicity Testing of Chemicals Japan, JETOC 2001)

- S. typhimurium TA1535, TA100, TA1537 and TA98, with and without metabolic activation (Ames test): negative (comp. OECD 471, BASF 1988)

- S. typhimurium TA 1535, TA 1537, TA98 and TA100, and E. coli WP2 uvrA, with and without metabolic activation (Ames test): negative (acc.Guidelines for Screening Mutagenicity Testing of Chemicals Japan,...

Link to relevant study records

Reference

Reference 1

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.

Reference 2

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.

Reference 3

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

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

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:

other: OECD Guideline 487 (Draft Proposal), 13 Dec 2007

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

The micronucleus assay is a method independent of the karyotype of the cells used for an indirect detection of damage of chromosomes or the mitotic apparatus. Micronuclei are formed either from acentric chromosome fragments as a result of a chromosome-breaking (clastogenic) effect or by entire chromosomes as a consequence of impairments of chromosome distribution in the course of mitosis (aneugenic effect).

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 ceII line has a high proliferation rate (doubling time of about 12 - 16 hours), a high plating efficiency (~ 90 %) as well as a stable karyotype (modal number of 22 chromosomes).
Stocks ofthe V79 cell line (1-ml portions) were maintained at -196 °C in liquid nitrogen using 7 % DMS0 in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for mycoplasma contamination, karyotype stability and plating efficiency (incl. vital staining).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male SD rats (treated i.p. with 500 mg/kg bw Aroclor 1254 five 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:

- Mixed population method:
24 hours exposure, 24 hours harvest time, without S9-mix: 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25.0 and 50.0 µl/ml;
4 hours exposure, 24 hours harvest time, with S-9 mix: 0, 0.78, 1.56, 3.125, 6.25, 12.5, 25.0 and 50.0 µl/ml
- Mitotic shake off method:
24 hours exposure, 27 hours preparation time, without S-9 mix: 0, 1.56, 3.156, 6.25, 12.5, 25.0, 50.0 and 75.0 µg/ml;
4 hours exposure, 27 hours preparation time, with S-9 mix: 0, 1.560, 3.125, 6.25, 12.5, 25.0, 50.0 and 75.0 µg/ml;

Vehicle / solvent:

In comparison to other commonly used vehicles (e.g. water, acetone), DMSO is the most suitable. Therefore DMSO was selected as the vehicle which had been demonstrated to be suitable in the V79 in vitro micronucleus assay and for which historical control data is available.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Pretests for dose selection:
- Mixed Population Method: (24 hours sampling time); tests with cultures exposed to a wide dose range, i.e. 0.5 - 2500 µg/ml culture medium both without S-9 mix (24 hours continuous treatment) and after adding a metabolizing system (pulse treatment of 4 hours); 50 µg/ml was (with and without S-9 mix) the top dose selected. Due to strong test substance precipitation which interferes with evaluation of cells higher doses could not be evaluated.
- Mitotic Shake Off Method: 75 µg/ml (with and without S-9 mix) was selected as top dose, because a strong test substance precipitation was found at higher concentration levels, which interferes with evaluation of cells.

Cell cycle time: ca. 13-14 hours under the selected culture conditions

Preparation of test cultures:
- Mixed Population Method:
The experimental procedure was carried out based an the method of Kalweit. et al. (Mut. Res. 439: 183-190, 1999). Logarithmically growing cultures (after the 6th passage) which were more than 50 % confluent were trypsinized (0.25 % trypsin solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Prior to trypsin treatment, the cells were rinsed once with 5 ml Ca-Mg-free HBSS. This process was stopped by adding MEM supplemented with 10 % FCS. A single ceII suspension was prepared, and about 5 ml MEM supplemented with 10 % FCS and containing about 30000 - 80000 cells were seeded in each chamber of Quadriperm dishes. Two chambers of a Quadriperm dish were used for one test culture. The Quadriperm dishes were incubated with 5 % CO2 at 37 °C and > 90 % humidity.
Treatment of test cultures:
Without S-9 mix: About 6 hours after seeding and incubating the cells, the medium was replaced by 4 ml fresh medium with FCS. The test article, suspended in 50 µI DMS0 was added to the culture medium. Concurrent negative and positive controls were tested. The cultures were incubated for 24 hours with 5% CO2 at 37°C and > 90 % humidity.
With S-9 mix: About 6 hours after seeding and incubating the cells, the medium was replaced by 3 ml of fresh medium (without FCS). The test article, suspended in 50 µI, was added to the culture medium along with 1 ml S-9 mix. Concurrent negative and positive controls were tested. After incubation (5 % CO2, 37 °C and > 90 % humidity) for 4 hours, the serum-free medium was replaced by 5 ml MEM supplemented with 10 % FCS after being rinsed twice with 5 ml HBSS. Subsequently, the Quadriperm dishes were incubated again with 5 % CO2 at 37 °C and > 90 % humidity for another 20 hours until the cells were harvested.

- Mitotic Shake 0ff Method:
The experimental procedure was carried out based on the method of Seelbach et al. (Toxicol. in Vitro 7: 185-193, 1993). Logarithmically growing cultures (after the 3rd passage) which were more than 50 % confluent were trypsinized (0.25% trypsin solution and Ca-Mg-free Hanks' balanced salt solution, HBSS). Prior to trypsin treatment, the celis were rinsed once with 5 ml Ca-Mg-free HBSS. This process was stopped by adding MEM supplemented with 10 % FCS. A single cell suspension was prepared, and about 25 ml MEM supplemented with 10 % FCS and containing about 2 x 10exp+6 cells were seeded in each culture flask. One flask was used per test culture. The flasks were incubated with 5 % CO2 at 37 °C and > 90 % humidity.
Treatment of test cultures:
Without S-9 mix: About 16 - 24 hours after seeding and incubating the cells, the medium was replaced by 20 ml fresh medium with FCS. The test article, suspended in 250 µI DMS0, was added to the culture medium. Concurrent negative and positive controls were tested. The cultures were incubated for 24 hours with 5 % CO2 at 37 °C and > 90 % humidity.
With S-9 mix: About 16 - 24 hours after seeding and incubating the cells, the medium was replaced by 15 ml of fresh medium without FCS. The test artcle, suspended in 250 µl DMS0, was added to the culture medium along with 5 ml S-9 mix. Concurrent negative and positive controls were tested. After incubation (5% CO2, 37 °C and > 90 % humidity) for 4 hours, the serum-free medium was replaced by 25 ml MEM supplemented with 10 % FCS after being rinsed twice with 10 ml HBSS. Subsequently, the flasks were incubated again with 5% CO2 at 37 °C and > 90 % humidity for another 20 hours until mitotic shake off.
Mitotic Shake 0ff:
24 hours following the treatment of the cells, the mitotic cells were shaken off the monolayer by hitting the flasks and were transferred into sterile centrifuge tubes. The cells were centrifuged at 100 x g for 5 minutes and afterwards the supernatant was removed and the ceII pellet was resuspended in 2 ml MEM with 10 % FCS. Approx. 0.5 ml of the cdl suspension-was transferred onto microscope slides in Quadriperm dishes (2 slides per test culture) and 2.5 ml of medium with 10 % FCS was added. The dishes were then incubated with 5 % CO2 at 37 °C and > 90 % humidity for 3 hours until harvesting.
CeII harvest and preparation of slides
The cells were prepared based on the methods described byKalweit et al. (Mut. Res. 439: 183-190, 1999). After incubation the culture medium was completely removed. For hypotonic treatment, 5 ml of a 1.5 % Sodium citrate solution at 37 °C was added for about 5 minutes. Following aspiration of the hypotonic solution, 5 ml of fixative (ethanol : glacial acetic acid Formaldehyde (37 %)/3: 1: 0.0125) which was at 4 °C was added for ca. 5 minutes and then removed and 5 ml of fresh fixative was added for at least another 5 minutes at room temperature for complete fixation. The sildes were taken out of the Quadriperm chambers, briefly allowed to drip and then rapidly passed through a Bunsen burner flame. The preparations were dried in the air and subsequently stained in Wrights solution (modified Mav-Gründwald solution) for ca. 3 minutes. After being rirised once in Titrisol pH 7.2, the slides were stained in a 2.6 % Giemsa solution (5.2 ml Giemsa, 195 ml Titrisol pH 7.2) for ca. 20 minutes. After being rirsed twice in Titrisol pH 7.2 and clarified in xylene, the preparations were mounted using Gorbit-Balsam.

Evaluation:
As a rule, 2000 cells from each dose group, each dose group consisting of 2 test cultures, were evaluated and the number of micronucleus-containing cells was recorded.
The analysis of micronuclei was carried out, observing the following criteria:
- the micronucleus consists of an area less than 1/3 of the area of the main nucleus
- the micronucleus and main nucleus retain the same staining
- the micronucleus is not linked to the main nucleus and is located within the cytoplasm of the cell
- for evaluation, only cells clearly surrounded by a nuclear membrane were scored.
Slides were coded before microscopic analysis. Cultures with few isolated cells were not analysed for micronuclei.
Since the absolute values shown have been rounded off but the calculations were made using the unedited values, deviations in the given relative values can occur.
- Mitotic index (Ml):
A mitotic Index based on 1000 cells/culture was determined for all test groups both in the Mixed Population Method and Mitotic Shake 0ff Method.
- Cell count
For the determination of cytotoxicity, additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the Mixed Population Method. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison with the concurrent vehicle control at the end of the culture period using a counting chamber.
- Cell morphology
About 3 - 4 hours after test substance treatment with S-9 mix and about 22 - 24 hours after treatment without S-9 mix, the cell morphology, which is an indication of attachment of the cells to the slides or flasks, was checked for each culture in all test groups using both modifications of the assays (with the exception of the positive controls).
- Fragmentation
During evaluation of 1000 cells/culture the occurance of fragmentation (fragmented nuclei or multinucleated cells and cells with > 6 micronuclei) was recorded.
Evaluation of the preparations:
Before analysis, the finished preparations were checked for the following
- possible reduction in the quality of the cells
- number of arialyzable cells
- amount of fragmented cells
The analyzable dose groups were determined based on the findings of this control check.
- Proliferal:ion Index (PI)
In addition to the evaluation of micronuclei and mitotic index in the Mixed Population Method, the proliferation index was determined as a measure of cytotoxicity. The PI, based on 1000 cells per culture (2000 cells per dose group), was determined for all test groups with or without S-9 mix. The number of clones (packs) consisting of 1 cell, 2, 4 or 8 cells was recorded and the Pl was calculated using the following formula:
PI = ((ncl-1) x 1 + (ncl-2) x 2 + (ncl-4) x 3 + (ncl-8) x 4) / total no. of clones
cl-1 = cell clone with 1 cell
cl-2 = cell clone with 2 cells
cl-3 = cell clone with 3 or 4 cells
cl-4 = cell clone with 5, 6, 7 or 8 cells

Controls:
- Vehicle control: The vehicle controls with and without S-9 mix only contained the vehicle for the test substance at the same concentration and volume used in the test culture.
- Positive controls:
The following positive control substances were used to demonstrate the sensitivity of the test method and the activity of the S-9 mix:
* Ethyl-Methane-Sulfonate (EMS): For the detection of clastogens without metabolic activation, 350 pg EMS (SIGMA, M-0880)/ml culture medium added in a volume of 1 ml (Mixed Population method) or 5 ml (Mitotic Shake 0ff method).
* Cyclophosphamide (CPP): For the detection of clastogens with metabolic activation, 2.5 pg CPP Endoxan®, ASTA MEDICA, Reg. Nr. E 432-1 )/ml culture medium added in a volume of 1 ml (Mixed Population method) or 5 ml (Mitotic Shake 0ff method).
The stability of EMS and CPP is well-defined under the selected culture conditions, since both positive control articles are well-established reference clastogens.

Evaluation criteria:

Acceptance criteria:
The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed, at least to a large extent, the identification and evaluation of a sufficient number of analyzable cells.
- The proportion of cells with micronuclei in negative control (vehicle control) cultures was within the normal range ofthe historical control data.
- The positive control chemicals (with and without S-9 mix) induced a significant increase in the number of cells with micronuclei.

Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible significant increase in the number of cells containing micronuclei.
- The proportion of micronucleus-containing cells exceeded both the concurrent negative control range and the negative historical control range.
A test substance is generally considered nongenotoxic in this test system if:
- There was no significant increase in the number of micronucleus-containing cells at any dose above concurrent negative control frequencies and within the historical control data.

Statistics:

Due to the clear negative finding, a statistical analysis was not carried out.

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

MIXED POPULATION METHOD
- Micronucleus Frequency:
An increase in the number of cells containing micronuclei was not observed either without S-9 mix or after the addition of a metabolizing system.
- Mitotic Index (MI):
The mitotic Index was based on 1000 cells per culture for the different test groups with and without metabolic activation. According to the results of the determination of the mitotic index, no suppression of the mitotic activity was observed under all experimental conditions.
- Proliferation Index (PI):
The proliferation Index is based on 1000 cells per culture (2000 cells per test group) for the different test groups without and with metabolic activation and allows the measurement of colony sizes. According to the results of the determination of the PI, no cytotoxic response was observed under any of the experimental conditions.
- CelI Count:
According to the results of the ceIl count, no growth inhibition was observed under all experimental conditions.
- CeII Morphology:
CeIl attachment was not influenced at any dose evaluated for micronuclei.
- Treatment Conditions:
Osmolality and pH values were not influenced by test substance treatment.
- Solubility:
Concentrations >10.0 µg/ml (without S-9 mix) or > 12.5 mg/ml (with S-9 mix) led to strong precipitation which interferes with evaluation of cells.

MITOTIC SHAKE 0FF METHOD
- Micronucleus Frequency:
An increase in the number of micronucleated cells was not found either without S-9 mix or after the addition of a metabolizing system.
- Mitotic Index:
The mitotic index was based on 1000 cells per culture for the different test groups with and without metabolic activation. According to the results of the determination af the mitotic index, no suppression of the mitotic activity was observed under any of the experimental conditions.
- CeIl Morphology:
Cell attachment was not influenced at any dose evaluated for micronuclei.
- Treatment Conditions:
Osmolality and pH values were not influenced by test substance treatment.
- Solubility:
Strong test substance precipitation in the vehicle was observed in all dose groups from about 6.25 µg/ml onward.

The increase in the frequencies of micronuclei induced by the positive control agents, EMS and CPP, cIearly demonstrated the sensitivity of the test method and of the metabolic activity of the S-9 mix employed.

Micronucleus frequency (absolute) for Mixed Population Method based on  
1000 cells per culture (2000 cells per test group; 24 hours mitotic shake off) 
observed are summarised below:
- without activation:
-----------------------------------------------------------
Test            /Dose    /mean from   /mean   /mean mitotic
substance       /(µg/ml) /2 cultures  /%      /index (abs.)
-----------------------------------------------------------
DMSO            /        /13.0        /1.3    /28

Positive        /350     /35.5        /3.55   /16

Test substance  /0.78    /8.5         /0.85   /28
                /1.56    /5.0         /0.5    /15
                /3.125   /8.0         /0.8    /19
                /6.25    /5.0         /0.5    /20
                /10.0    /8.0         /0.8    /16
-----------------------------------------------------------

- with activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /17.0        /1.7    /21

Positive        /350     /124         /12.4   /30

Test substance  /0.78    /7.0         /0.7    /18
                /1.56    /6.0         /0.6    /16
                /3.125   /8.0         /0.8    /23
                /6.25    /5.5         /0.55   /18
                /10.0    /7.5         /0.75   /21
----------------------------------------------------------

Micronucleus frequency (absolute) for Mitotic Shake off Method based on 1000 
cells per culture (2000 cells per test group; 24 hours mitotic shake off) 
observed are summarised below:
- without activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /4.5         /0.45  /28

Positive        /350     /46.5        /4.65  /16

Test substance  /6.25    /2.0         /0.20  /20
                /12.5    /9.5         /0.95  /24
                /25.0    /5.5         /0.55  /24
                /50.0    /7.0         /0.70  /14
                /75.0    /7.5         /0.75  /18
----------------------------------------------------------

- with activation:
----------------------------------------------------------
Test            /Dose    /mean from   /mean  /mean mitotic
substance       /(µg/ml) /2 cultures  /%     /index (abs.)
----------------------------------------------------------
DMSO            /        /3.5         /0.35  /8

Positive        /2.50    /57          /5.70  /20

Test substance  /1.56    /4.5         /0.45  /12
                /3.125   /6.5         /0.65  /12
                /6.25    /2.0         /0.20  /9
                /12.50   /4.5         /0.45  /19
                /25.0    /2.5         /0.25  /10
----------------------------------------------------------

Conclusions:

Thus, under the experimental conditions of this assay, Heliogen Gruen 8730 Pulver is considered not to be a chromosome-damaging (clastogenic) agent nor does it induce numerical chromosomal aberrations (aneugenic activity) in-vitro in V79 cells.

Reference 5

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:

other: Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473

Deviations:

no

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

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:

Rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

without S9 mix: 9.77, 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate (S. typhimurium TA100, TA1535, TA98 and TA1537)
without S9 mix: 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2 uvrA)
with S9 mix: 156, 313, 625, 1250, 2500 and 5000 µg/plate (S. typhimurium TA100, TA1535, TA98 and TA1537)
with S9 mix: 313, 625, 1250, 2500 and 5000 µg/plate (E. coli WP2 uvrA)

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

A Preincubation test was conducted.
Plates per test: 3
Number of replicates: 2

POSITIVE CONTROLS:
without S9-mix: AF-2 (TA 100, WP2, TA98), sodium azide (TA 1535) and 9-aminoacridine (all strains)
with S9-mix: 2-aminoanthracene (all strains)

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

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:

cytotoxicity

Remarks:

see details in "additional information on results"

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:

MUTAGENICITY:
No increase in the number of his+ revertants was observed, with or without the addition of S9 mix in all S. typhimurium strains tested (TA102, TA100, TA98, TA97) as well as in E. coli WP2 uvr A.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

CYTOTOXICITY:
Toxicity was observed at 313 µg/plate or more (TA100 and TA1535) and at 625 µg/plate (TA98 and TA1537) without metabolic activation, and at 5000 µg/plate (TA100, TA1535, TA98 and TA1537) with metabolic activation.

The maximum revertants/plate occurring at the non-cytotoxic (lowest) 
concentrations of the test substance were the following:
- First experiment
--------------------------------------------------------------------------------------
Strain  /Dose   /Maximum revertants /Positive controls   /Cytotoxic concentration(µg)
        /(µg)   /-S-9     /+S-9     /-S-9      /+S-9     /-S-9     /+S-9
--------------------------------------------------------------------------------------
TA-100  /0      /97+-4    /97+-4    /478+-27   /1154+-62 />=313    /5000
        /156    /105+-16  /         /          /         /         /
        /313    /         /100+-3   /          /         /         /

TA-1535 /0      /9+-1     /11+-4    /440+-10   /222+-37  />=313    /5000
        /9.77   /11+-4    /         /          /         /         /
        /2500   /         /11+-4    /          /         /         /

WP2 uvrA/0      /23+-6    /31+-8    /682+-30   /1435+-66 /-        /-
        /313    /25+-5    /         /          /         /         /
        /1250   /         /33+-7    /          /         /         /

TA-98   /0      /15+-2    /28+-2    /467+-44   /526+-30  /625      /5000
        /19.5   /22+-2    /         /          /         /         /
        /625    /         /32+-5    /          /         /         /

TA-1537 /0      /9+-1     /21+-2    /387+-88   /177+-25  /625      /5000
        /19.5   /11+-2    /         /          /         /         /
        /2500   /         /21+-3    /          /         /         /
--------------------------------------------------------------------------------------

- Second experiment
--------------------------------------------------------------------------------------
Strain  /Dose   /Maximum revertants /Positive controls   /Cytotoxic concentration (µg)
        /(µg)   /-S-9     /+S-9     /-S-9      /+S-9     /-S-9     /+S-9
--------------------------------------------------------------------------------------
TA-100  /0      /100+-10  /97+-7    /690+-41   /1480+-20 />=313    /5000
        /39.1   /115+-16  /         /          /         /         /
        /2500   /         /103+-6   /          /         /         /

TA-1535 /0      /7+-2     /9+-2     /411+-63   /262+-16  />=313    /5000
        /39.15  /9+-4     /         /          /         /         /
        /625    /         /11+-3    /          /         /         /

WP2 uvrA/0      /28+-1    /33+-4    /861+-67   /1313+-149/-        /-
        /625    /28+-3    /         /          /         /         /
        /625    /         /31+-4    /          /         /         /

TA-98   /0      /17+-4    /36+-3    /579+-24   /579+-18  /625      /5000
        /156    /         /28+-2    /          /         /         /
        /313    /24+-5    /         /          /         /         /

TA-1537 /0      /11+-2    /11+-1    /545+-145  /224+-15  /625      /5000
        /156    /16+-3    /         /          /         /         /
        /1250   /         /13+-2    /          /         /         /
--------------------------------------------------------------------------------------

Conclusions:

Based on the results the test substance is not a mutagenic in the Ames test under the experimental conditions of this study chosen.

Reference 6

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

other: Guidelines for Screening Mutagenicity Testing of Chemicals (Chemical Substances Control Law of Japan) and OECD Test Guideline 473

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosomal aberrations in cultured mammalian cells. Structural aberrations may be of chromosome or chromatid type.

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- without S9-mix (6 hour short-term treatment): 0, 125, 250, 500 µg/ml
- with S9-mix (6 hour short-term treatment): 0, 125, 250, 500 µg/ml
- without S9-mix (24 hour continuous treatment): 0, 125, 250, 500 µg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

200 cells were analyzed in each group for chromatid braks, chromatid exchange, chromosome break, chromosome exchange (dicentric and ring).
Judgement was done on the basis of criteria of Ishidate et al. 1987.

Species / strain:

Chinese hamster lung (CHL/IU)

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 test substance did not induce chomosomal aberrations or polyploidy with or without metabolic activation.
At the end of the treatment period at 6 h and at 24 h, the test substance foamed membrane-like sheet onto the medium and precipitation of the test substance in the medium was also observed.
At the end of the treatment period at 24 h the colour of the medium was changed to green.

Chromosomal analysis of the treated cells :
- after 6 hours treatment (without S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /3                /0            /0.0
Substance  /125    /101          /200      /0                /0            /1.0
           /250    /123          /200      /2                /1            /0.0
           /500    /110          /200      /2                /0            /1.0

MMC        /0.1    /87           /200      /90               /0            /0.0
-------------------------------------------------------------------------- ------------

- after 6 hours treatment (with S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /1                /0            /0.0
Substance  /125    /97           /200      /1                /1            /0.5
           /250    /62           /200      /1                /0            /0.0
           /500    /61           /200      /0                /0            /0.5

BP         /20     /75           /200      /199              /1            /0.0
-------------------------------------------------------------------------- ------------

- after 24 hours treatment (without S-9 mix)
-------------------------------------------------------------------------- ------------
                                 /Number of cells
Treatment  /Dose    /Cell growth ----------------------------------------  /Polyploid
           /(µg/ml) /index       /analysed /with aberrations /with gaps    /(%)
-------------------------------------------------------------------------- ------------
Test       /0      /100          /200      /2                /0            /0.0
Substance  /125    /94           /200      /1                /0            /0.0
           /250    /94           /200      /1                /0            /0.5
           /500    /90           /200      /0                /0            /0.5

MMC        /0.03   /68           /200      /72               /5            /0.0
-------------------------------------------------------------------------- ------------

Conclusions:

No significant increases in the incidence of the structural chromosomal aberrations or polyploidy was seen in the cells under the experimental conditions of the study chosen.

Reference 7

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

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:
Standard plate test with and without S9-mix (all strains): 0, 20, 100, 500, 2500 and 5000 µg per plate
3rd experiment:
Preincubation test with and without S9-mix (all strains): 0, 4, 20, 100, 500 and 2500 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:

cytotoxicity

Remarks:

Slight decrease in number of revertants depending on strain and test conditions (>500-2500 µg/plate onward).

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:

cytotoxicity

Remarks:

Slight decrease in number of revertants depending on strain and test conditions (>500-2500 µg/plate onward).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY:
A slight decrease in the number of revertants was occasionally observed depending on the strain and test conditions from about 500 µg - 2500 µg/plate onward.

SOLUBILITY:
Test substance precipitation was found from about 20 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	18 ± 2	18 ± 2
	Test substance	19 ± 4 [20]	17 ± 2 [20]
	Positive Control	709 ± 35 [5; MNNG]	174 ± 6 [2.5; 2-AA]
S. typhimurium TA100	DMSO	135 ± 17	129 ± 14
	Test substance	128 ± 4 [20]	137 ± 5 [100]
	Positive Control	772 ± 77 [5; MNNG]	788 ± 42 [2.5; 2-AA]
S. typhimurium TA98	DMSO	31 ± 2	35 ± 6
	Test substance	26 ± 4 [20]	31 ± 3 [20]
	Positive Control	953 ± 30 [10; NOPD]	878 ± 23 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	11 ± 3	12 ± 3
	Test substance	10 ± 2 [20]	11 ± 2 [100]
	Positive Control	746 ± 24 [100; AAC]	131 ± 14 [2.5; 2-AA]
E. coli WP2 uvrA	DMSO	25 ± 2	27 ± 3
	Test substance	25 ± 1 [100]	30 ± 4 [20]
	Positive Control	1128 ± 25 [5; 4-NQO]	204 ± 8[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	21 ± 2	21 ± 2
	Test substance	18 ± 2 [4]	17 ± 2 [4]
	Positive Control	1083 ± 46 [5; MNNG]	112 ± 8 [2.5; 2-AA]
S. typhimurium TA100	DMSO	137 ± 15	145 ± 4
	Test substance	136 ± 13 [500]	143 ± 14 [500]
	Positive Control	1066 ± 64 [5; MNNG]	687 ± 49 [2.5; 2-AA]
S. typhimurium TA98	DMSO	26 ± 3	42 ± 6
	Test substance	26 ± 5 [100]	35 ± 1 [4]
	Positive Control	943 ± 12 [10; NOPD]	739 ± 21 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	12 ± 3	12 ± 1
	Test substance	9 ± 2 [20]	11 ± 1 [20]
	Positive Control	559 ± 59 [100; AAC]	113 ± 15 [2.5; 2-AA]
E. coli WP2 uvrA	DMSO	32 ± 2	37 ± 2
	Test substance	32 ± 3 [4]	28 ± 1 [4]
	Positive Control	782 ± 60 [5; 4-NQO]	213 ± 11 [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.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

Principles of method if other than guideline:

The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA1535, TA1537, TA98 and TA100. However, the mutagenic potential of the test substance was not tested in an additional E. coli strain or in S. typhimurium TA102, as recommended in the latest version of OECD guideline 471 (July 1997).

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors (MgCl2, KCl, glucose-6-phosphate, NADP, phosphate buffer pH 7.4).

Test concentrations with justification for top dose:

- Standard plate test:
1st experiment:
0, 20, 100, 500, 2500 and 5000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537), with and without metabolic activation.
2nd experiment:
0, 100, 500, 2500, 5000 and 10000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537) with and without metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see details on test system

Details on test system and experimental conditions:

METHOD OF APPLICATION:
A standard plate was conducted, with and without metabolic activation (S9-mix). Each test was conducted in triplicates.

STANDARD PLATE TEST:
The experimental procedure is based on the method of Ames et al. (Mut. Res. 31: 347-364, 1975):
Test tubes containing 2 ml portions of soft agar which consists of 100 ml agar (0.6 % agar + 0.6 % 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 45 °C and the remaining components are added in the following order:
0.1 ml test solution
0.1 ml bacterial suspension
0.5 ml S-9 mix (in tests with metabolic activation)
or
0.5 ml phosphate buffer (in tests without metabolic activation)
After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within approx. 30 seconds.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (solvent control with DMSO) is carried out for each tester strain in order to determine the spontaneous mutation rate.
- Positive controls:
The following positive control substances are used to check tIhe mutability of the bacteria and the activity of the S-9 mix:
with S-9 mix: 10 µg 2-aminoanthracene (dissolved in DMSO) for the strains TA 100, TA 98, TA 1537 and TA 1535.
without S-9 mix: 5 µg N-methyl-N´-nitro-N-nitrosoguanidine (MNNG) (dissolved in DMSO) for the strains TA 100 and TA 1535; 10 µg 4-nitro-o-phenylendiamine (dissolved in DMSO) for the strain TA 98; 100 µg 9-aminoacridine chloride monohydrate (dissolved in DMSO) for the strain TA 1537.

TITER DETERMINATION:
In general, the titer is determined only in the experiments with S-9 mix both without test substance (solvent only) and after adding the two highest doses of test substance. For this purpose, 0.1 ml of the overnight cultures is diluted to 1 x 10exp-6 in each case. Test tubes containing 2 ml portions of soft agar containing maximal amino acid solution (5 mM histidine + 0.5 mM biotin) are kept in a water bath at 45 °C, and the remaining components are added in the following order:
0.1 ml solvent (without and with test substance)
0.1 ml bacterial suspension (dilution: 1 x 10exp-6)
0.5 ml S-9 mix
After mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds. After incubation at 37°C for 48 hours in the dark, the bacterial colonies are counted.

OTHER EXAMINATIONS:
The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character, UV sensitivity, ampicillin resistance. Histidine auxotrophy is automatically checked in each experiment via the spontaneous rate.

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

in the standard plate test

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

no bacteriotoxic effect was observed

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

MUTAGENICITY:
No increase in the number of his+ revertants was observed in the standard plate test, with or without the addition of S9 mix in all S. typhimurium strains tested (TA1535, TA100, TA1537, TA98).
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

SOLUBILITY:
The test substance was incompletely soluble in DMSO from about 500 µg/plate onward.

TOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed in the standard plate test with and without S9-mix.

Maximum revertants/plate and corresponding test concentrations in the preincubation test:

1st experiment:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	14 ± 2	16 ± 4
	Test substance	28 ± 22 [5000]	15 ± 1 [20]
	Positive Control	1983 ± 231 [5; MNNG]	526 ± 22 [10; 2 -AA]
S. typhimurium TA100	DMSO	115 ± 20	97 ± 8
	Test substance	104 ± 15 [2500]	116 ± 11 [100]
	Positive Control	1717 ± 35 [5; MNNG]	1905 ±  65 [10; 2 -AA]
S. typhimurium TA1537	DMSO	6 ± 2	7 ± 3
	Test substance	8 ± 2 [2500]	10 ± 3 [20]
	Positive Control	654 ± 235 [10; NOPD]	141 ± 20 [10; 2 -AA]
S. typhimurium TA98	DMSO	17 ± 2	33 ± 7
	Test substance	23 ± 1 [500]	37 ± 4 [20]
	Positive Control	789 ± 200 [100; AAC]	1537 ± 93 [10; 2 -AA]
2nd experiment
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	15 ± 2	18 ± 3
	Test substance	14 ± 2 [2500]	18 ± 2 [500]
	Positive Control	1260 ± 122 [5; MNNG]	236 ± 26  [10; 2 -AA]
S. typhimurium TA100	DMSO	120 ± 5	118 ± 14
	Test substance	116 ± 14 [100]	117 ± 12 [500]
	Positive Control	1407 ± 70 [5; MNNG]	1433 ± 61 [10; 2 -AA]
S. typhimurium TA1537	DMSO	9 ± 2	10 ± 2
	Test substance	10 ± 3 [5000]	11 ± 4 [500]
	Positive Control	557 ± 100 [10; NOPD]	131 ± 18 [10; 2 -AA]
S. typhimurium TA98	DMSO	25 ± 1	37 ± 4
	Test substance	24 ± 3 [100]	39 ± 7 [2500]
	Positive Control	665 ± 88 [100; AAC]	743 ± 95 [10; 2 -AA]
2-AA = 2-aminoanthracene
MNNG = N-methyl-N'-nitro-N-nitrosoguanidine
NOPD = 4-nitro-o-phenylenediamine
AAC = 9-aminoacridine

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.

Reference 9

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

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:

yes

Remarks:

no metabolic activation (investigated in separate study), no independent repeat experiment.

GLP compliance:

yes (incl. QA statement)

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:

- Purity: 95%
- Substance type: pigment
- 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:

mammalian cell line, other: Human Fibroblasts CRL 1121

Details on mammalian cell type (if applicable):

- Type and identity of media: DULBECCO's Minimal Essential Medium containing 10% foetal bovine serum, 100 U/ml penicillin, 100 ug/ml streptomycin and 2.5 ug/ml amphotericin
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (cytoplasmic labelling with 3H-thymidine in the autoradiographs would be strongly indicative for infection)
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
- Passage No. 14

Metabolic activation:

without

Test concentrations with justification for top dose:

60.0, 12.0, 2.40 and 0.48 ug/ml.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

5 uM

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24h
- Exposure duration: 5h

STAIN: trypane blue for cytotoxicity assay, 3H-thymidine, autoradiography (6 days exposure) for DNA-repair assay; The autoradiographs are stained with hematoxylin-eosine.

PROPERTY

NUMBER OF REPLICATIONS: 3

NUMBER OF CELLS EVALUATED: 150

DETERMINATION OF CYTOTOXICITY
- Method: trypan blue (0.2%) staining

OTHER:
The slides are coded prior counting. From each of the treatment groups and from the positive and the negative controls 150 nuclei
in altogether three slides (50 cells/slide) are scored, the number of silver grains counted, the mean values and the standard deviations calculated. Counting of silver grains over the nuclei of the fibroblasts is carried out with the aid of an electronic counter (ARTEK Model 982) attached to a microscope (ZEISS) at a magnification of 2000x, using an objective 100x and a projective 10x.
The entry of cells into S-phase does not need to be blocked by any method, because experience has shown, that cells in the replicative DNA-synthesis-phase are easily visible in autoradiographic measurement, i.e. the nucleus is replete with silver grains (>120 silver grains/nucleus). These cells are excluded from the determination of the silver grains/nucleus count.
The background in the autoradiographs (outside the cells) is determined in cell-free areas microscopically.

Evaluation criteria:

The substance is generally considered to be active in the DNArepair test if one of the following conditions are 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.

Assay acceptance criteria:
- The results of the experiments should not be influenced by a technical error, contamination or a recognized artifact.
- The labelling in the vehicle control cultures should not exceed an average of five total grains/nucleus, or the number of the vehicle control nuclei with more than five silver grains should not exceed 10%.
- The positive control should fulfil all criteria set up 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 analyzed for nuclear grain counts.
- The highest analyzed concentration should approach an excessive toxicity (defined in the toxicity test), or result from test material insolubility, or be at least 100 mg/ml.

Statistics:

Duncan's multiple range test (Biometrics 31. 339-359 (1975))

Species / strain:

mammalian cell line, other: human fibroblast, line CRL 1521

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Background determination
Treatment group	concentration	Silver grain per nucleus-equivalent area
control (medium)		0.30
control (Vehicle)		0.15
positive control 4NQ	5 µM	0.4
Test substance	60.0 µg/ml	0.3
Test substance	12.0 µg/ml	0.2
Test substance	2.4 µg/ml	0.2
Test substance	0.48 µg/ml	0.4

Treatment group	concentration	Silver grain per nucleus-equivalent area ± SD
control (medium)	n.a.	1.20 ± 1.03
control (Vehicle)	n.a.	1.17 ± 1.04
positive control 4NQ	5 µM	34.3 ± 12.61
Test substance	60.0 µg/ml	1.63 ± 1.16
Test substance	12.0 µg/ml	1.47 ± 1.23
Test substance	2.4 µg/ml	1.27 ± 1.07
Test substance	0.48 µg/ml	1.31 ± 1.03

Conclusions:

negative without metabolic activation

Copper phthalocyanine does not induce DNA-repair activiity in a human fibroblast cell line that is exposed for 5h without additional metabolic activation.

Executive summary:

Based on dose-range-finder experiments, 60.0, 12.0, 2.40 and 0.48 ug/mlwere used as test concentrations. The highest dose resulted in visible precipitation. DMSO was used as vehicle. The incubation period with the test material was five hours. DNA repair activity was detected by incorporation of 3H-thymidine, which was visualized by autoradiography.

In the experiments performed, comparison of the mean number of silver grains per nucleus in the negative controls and in the cultures treated with the various concentrations of Pigment Blue 15 revealed no marked deviations. By contrast, "positive control" experiments with 4NQ0 (5uM)yielded a mean value of 34.3 silver grains per nucleus. This value differs greatly from the two negative controls, by factors of 28.6 and 29.3.

Since no independent repeat experiment was performed, this study is assigned a validity score of 2. The study report contains a Quality Unit statement.

Reference 10

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Principles of method if other than guideline:

A positive control (benzo(a)pyrene) was used, however, the results of the positive control were not listed.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosomal aberrations in cultured mammalian cells. Structural aberrations may be of two types, chromosome or chromatid.

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from the liver of rats, treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

0, 0.75, 1.5, 3 mg/ml with and without metabolic activation.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

A Chromosomal Aberration Assay was conducted according to the Guideline for Screening Mutagenicity Testing of Chemicals (Japan).
4 different experimental protocols were conducted, as recommended in the Japanese Guideline:
- Continuous treatment for 24 h without S9-mix (24/24 -S9).
- Continuous treatment for 48 h without S9-mix (24/24 -S9).
- Pulse treatment for 6 h without S9-mix followed by harvesting at 24 h (6/24 -S9).
- Pulse treatment for 6 h with S9-mix followed by harvesting at 24 h (6/24 +S9).

All chromosome aberrations observed (chromatid and chromosome gap, chromatid break, chromatid exchange, chromosome break, exchange) were recorded as number of the respective effect per 100 cells (equates to %).

Positive control: Benzo(a)pyren

Plates per dose: 1

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: with metabolic activation: > 3 mg/ml, without metabolic activation: > 3 mg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

None of dose groups induced any chromosomal aberrations or polyploidy in CHL cells with or without S9-mix.

Table 1: Results of Chromosomal Aberration test (continuous treatment for 24 h and 48 h without S9-mix):

	Treatment time (h)	Concentration (mg/ml)	Number of cells	Polyploid	No. of chromosomal aberrations						No. of cells with Chr. Ab. Incl. Gaps
					Chromatid type			Chromosome type		others
					g	ctb	cte	csb	cse
DMSO	24	0	100	1	1	0	0	0	0	0	1
	48	0	100	1	1	0	0	0	0	0	1
Test substance	24	0.75	100	1	1	0	0	0	0	0	1
	24	1.5	100	3	0	1	0	0	0	0	1
	24	3.0	100	2	1	0	0	0	0	0	1
	48	0.75	100	2	0	0	0	0	0	0	0
	48	1.5	100	1	1	1	0	0	0	0	2
	48	3.0	100	2	0	0	0	0	0	0	0

Table 2: Results of Chromosomal Aberration test (pulse treatment for 6 h with and without S9-mix followed by harvesting at 24 h):

	S9-mix	Concentration (mg/ml)	Number of cells	Polyploid	Chromosomal aberration (%)						Number of cells with Chr. Ab. Incl. Gaps
					Chromatid type			Chromosome type		others
					g	ctb	cte	csb	cse
DMSO	-	0	100	0	0	0	0	0	0	0	0
	+	0	100	0	1	0	0	0	0	0	0
Test substance	-	0.75	100	1	3	0	0	0	0	0	3
	-	1.5	100	0	0	0	0	0	0	0	0
	-	3.0	100	0	0	0	0	0	0	0	0
	+	0.75	100	0	0	0	0	0	0	0	0
	+	1.5	100	0	0	0	0	0	0	0	0
	+	3.0	100	2	0	0	0	1	0	0	1

Abbreviations in table 1 and table 2:

g = chromatid and chromosome gap

ctb = chromatid break

cte = chromatid exchange

csb = chromosome break

cse = exchange

Conclusions:

According to the results of the present study, the tested substance was not mutagenic in the Chromosomal Aberration test under the experimental conditions chosen here.

Reference 11

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

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-mix from rat liver, induced with phenobarbital and 5,6.benzoflavone

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500 and 5000 µg/plate with or without S9-mix

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see "Details on test system and conditions"

Details on test system and experimental conditions:

A Preincubation test was conducted.
Plates per test: 3
Number of replicates: 2

POSITIVE CONTROLS:
without S9-mix: AF-2 (TA 100, WP2, TA98), sodium azide (TA 1535) and 9-aminoacridine (TA1535)
with S9-mix: 2-aminoanthracene (all strains)

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.

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:

MUTAGENICITY:
No increase in the number of his+ revertants was observed, with or without the addition of S9 mix in all S. typhimurium strains tested (TA102, TA100, TA98, TA97) as well as in E. coli WP2 uvr A.
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

CYTOTOXICITY:
No toxicity was observed up to a concentration of 5000 µg/plate withor without metabolic activation.

Conclusions:

Based on the results the test substance is not a mutagenic in the Ames test under the experimental conditions of this study chosen.

Reference 12

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosomal aberrations in cultured mammalian cells. Structural aberrations may be of chromosome or chromatid type.

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver, induced with phenobarbital and 5,6-benzoflavone

Test concentrations with justification for top dose:

- without S9-mix (6 hour short-term treatment): 0, 1300, 2500, 5000 µg/ml
- with S9-mix (6 hour short-term treatment): 0, 1300, 2500, 5000 µg/ml
- without S9-mix (24 hour continuous treatment): 0, 300, 600, 1200 µg/ml

Vehicle / solvent:

0.5 % Carboxymethylcellulose sodium

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:

No data given.

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXICITY:
The test substance did not induce chomosomal aberrations or polyploidy with or without metabolic activation.

Conclusions:

No significant increases in the incidence of the structural chromosomal aberrations or polyploidy was seen in the cells under the experimental conditions
of the study chosen.

Reference 13

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)

Remarks:

testing lab.

Type of assay:

bacterial reverse mutation assay

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 (all strains): 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 500 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.

Table 1: Maximum revertants/plate and corresponding test concentrations in the standard 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	20 ± 2
	Test substance	20 ± 2 [2500]	21 ± 1 [2500]
	Positive Control	655 ± 61 [5; MNNG]	206 ± 14 [2.5; 2-AA]
S. typhimurium TA100	DMSO	106 ± 2	105 ± 4
	Test substance	118 ± 5 [2500]	130 ± 19 [2500]
	Positive Control	737 ± 65 [5; MNNG]	1386 ± 116 [2.5; 2-AA]
S. typhimurium TA98	DMSO	30 ± 4	40 ± 4
	Test substance	25 ± 6 [500]	57 ± 4 [5000]
	Positive Control	836 ± 25 [10; NOPD]	960 ± 33 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	11 ± 2	12 ± 2
	Test substance	11 ± 1 [20]	12 ± 3 [2500]
	Positive Control	617 ± 13 [100; AAC]	168 ± 14 [2.5; 2-AA]
E. coli WP2 uvrA	DMSO	40 ± 1	52 ± 2
	Test substance	42 ± 6 [20]	52 ± 5 [2500]
	Positive Control	892 ± 21 [5; 4-NQO]	401 ± 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
Table 2: Maximum revertants/plate and corresponding test concentrations in the preincubation test:
Strain	Tested compound	Maximum revertants/plate [corresponding dose unit in µg/plate]
		without S9-mix	with S9-mix
S. typhimurium TA1535	DMSO	19 ± 1	20 ± 1
	Test substance	17 ± 1 [100]	19 ± 1 [20]
	Positive Control	1307 ± 225 [5; MNNG]	215 ± 15 [2.5; 2-AA]
S. typhimurium TA100	DMSO	110 ± 12	111 ± 6
	Test substance	108 ± 3 [20]	106 ± 2 [20]
	Positive Control	967 ± 26 [5; MNNG]	815 ± 65 [2.5; 2-AA]
S. typhimurium TA98	DMSO	35 ± 2	37 ± 4
	Test substance	32 ± 3 [20]	36 ± 5 [20]
	Positive Control	1265 ± 145 [10; NOPD]	861 ± 29 [2.5; 2-AA]
S. typhimurium TA1537	DMSO	8 ± 2	10 ± 2
	Test substance	9 ± 2 [100]	10 ± 3 [500]
	Positive Control	581 ± 51 [100; AAC]	95 ± 4 [2.5; 2-AA]
E. coli WP2 uvrA	DMSO	33 ± 1	29 ± 3
	Test substance	38 ± 3 [100]	27 ± 6 [5000]
	Positive Control	665 ± 10 [5; 4-NQO]	206 ± 10 [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:

Interpretation of results: negative
According to the results of the present study, the test substance was not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 14

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

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:

not specified

Type of assay:

bacterial reverse mutation assay

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:

liver S9 mix from rats treated with Arochlor 1254

Test concentrations with justification for top dose:

0, 312.5, 625, 1250, 2500 and 5000 μg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Evaluation criteria:

For a substance to be considered positive in this test system, it should have induced a dose-related and statistically(S) significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the 59 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results then a third experiment may be
used to confir~· the correct response. Tob~ considered negative the number of .induced revertants compared to spontaneous revertants should be less than twofold at each dose level amployed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 μg/plate. In this case the limiting factor was the maximum recommended
dose.

Statistics:

not needed

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxicity was exhibited to any of the strains of bacteria used. A precipitate was observed at and above 2500 μg/plate, this did not interfere
with the scoring of revertant colonies.

Experiment 1, without S9 mix
Test substance concentration per plate	TA100		TA1535		WP2uvrA·		TA98		TA1537
0	103	_(113)	19	(17)	24	(34)	22	(22)	1 1	(14)
	129		12		40		19		1 5
	108	13.8	20	4.4	37	8.5	25	3.0	15	2.3
8	106 94	(104)	11 20	(1 5)	34 41	(37)	16 20	(16)	1 3 13	(14)
	113	9.6	1 5	4.5	37	3.5	1 1	4.5	16	1.7
40	117 104	(108)	17 16	(15)	38 41	(38)	25 19	(21 )	9 1 1	(10)
	102	8.1	13	2.1	34	3.5	19	3.5	1 1	1.2
200	123 1 12	(110)	20 13	(15)	50 28	(40)	1 5 17	(18)	1 5 12	(14)
	95	14.1	13		42	11.1	22	3.6	16	2.1
1000	107	(105)	11	(10)	32	(30)	22	(21 )	16	(13)
	1 10		6		28		24		13
	98	6-2	13	3.6	29	2.1	16	4.2	9	3.5
5000	99P 124P	(107)	13P 13P	(14)	25P 24P	(28)	13P 19P	(17)	8P 1 1p	(10)
	99P	14.4	17P	2.3	36P	6.7	19P	3.5	1 2P	2.1
Positive control	ENNG		ENNG		ENNG		4NQO		9AA
Concentration (ug/plate)	3		5		2		0.2		80
	579		197		203		142		416
	594	(550)	173	(182)	190	(192)	123	(132)	342	(378)
	476	64.2	176	13.1	184	9.7	130	9.6	375	37.1

P = precipitation

Conclusions:

Interpretation of results: 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.

CAS No. 147-14-8:

- Chromosomal aberration in Chinese hamster bone marrow (in-vivo Nucleus Anomaly Test in Somatic Interphase Nuclei of Chinese Hamster): negative (CibaGeigy 1986, Val. 2)

- Gene mutation in-vivo, mouse (Mouse Spot Test): negative (Ciba Geigy 1986, Val. 2)

Link to relevant study records

Reference

Reference 1

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

Reference 2

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

CAS No. 147-14-8:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro.

Gene mutation in bacteria

Copper phthalocyanine was not mutagenic in a standard plate Ames test with and without metabolic activation (tested up to 10000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98 and TA100; metabolic activation: S9 fraction from the liver of male Sprague-Dawley rats, treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice and mixed with a series of cofactors. Cytotoxicity was not observed with or without metabolic activation.

In another Ames test a pre-incubation assay was conducted with and without metabolic activation (tested up to 5000μg/plate in Salmonella typhimurium TA100, TA 102, TA 98 and TA97; metabolic activation: S9 fraction from the liver of rats, treated with KC-400, equivalent to PCB), copper phthalocyanine was also not mutagenic (JETOC 1995). No cytotoxicity was observed.

Several other studies provided also negative results for copper phthalocyanine in the Ames test (Hayatsu 1983, Val. 4; Manandhar 1982, Val. 4). In another Ames test, a positive result was obtained. However, the mutagenicity test was conducted in two S. typhimurium strains (TA1535 and TA1538) only. No data were available in terms of cytotoxicity and validity of positive, solvent and negative controls (Milvy 1978, Val. 3).

Gene mutation in mammalian cells

The test substance was negative for genotoxicity in a mouse lymphoma assay using L5178Y cells at levels up to 1000 µg/plate (with and without metabolic activation) (Manandhar, Val. 4).

Cytogenicity in mammalian cells

CHL cells were used in an in-vitro chromosomal aberration test (acc. Japanese Guidelines for Screening Mutagenicity Testing of Chemicals, JETOC 1995), with and without metabolic activation. The test substance was found to be negative for causing cytogenicity at dose levels from 750 µg/ml up to 3000 µg/ml. Cytotoxicity was observed at > 3000 µg/ml.

Genome mutation in mammalian cells

A cell transformation test using C3H/10T1/2 was found to be negative for causing genome mutations at dose levels from 10 µg/plate up to 1000 µg/plate (Manandhar 1982, Val. 4).

DNA damage and/or repair

An UDS assay with rat hepatocytes was found to be negative for DNA damage/or repair at dose levels from 10 µg/plate up to 1000 µg/plate (Manandhar 1982, Val. 4).

 

It should be noted that there were several weakly positive Ames test results and one weakly positive HPRT test, when the test material TK 13143 (crude copper phthalocyanine) was investigated for mutagenic effects in bacteria as well as in V79 cells (Ciba-Geigy 1986 (3x) and 1988 (1x), all Val. 3). However, today it is known that these crude copper phthalocyanines were manufactured by using nitrobenzene and chlorobenzene as solvents in the past. Therefore the test material contained residues of these solvents and these impurities were considered to be responsible for the weakly mutagenic effects observed in-vitro. Meanwhile this manufacturing process was replaced by other manufacturing processes which all use alternative solvents.

 

GENOTOXICITY IN-VIVO:

Valid experimental data were also available to assess the genetic toxicity in-vivo.

Crude Copper Phthalocyanine was administered by gavage to Chinese hamsters (Cricetulus griseus) of either sex. Treatment consisted of one daily dose of 1250, 2500 or 5000 mg/kg on each of two consecutive days. The animals were sacrificed 24 h after the second application. From the bone marrow smears were made. The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo (Ciba Geigy 1986, Val. 2). The bone marrow smears from animals treated with the various doses of the test material showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponded to the frequency observed in the control group. By contrast, a "positive control" experiment with cyclophosphamide (128 mg/kg bw) yielded 9.48 % cells with anomalies of nuclei. This is significantly different from the controls (0.1 %) treated with the vehicle (0.5 % CMC) alone.

In a second in-vivo experiment, crude copper phthalocyanine was administered in a single intraperitoneal injection to pregnant female mice (C57 Bl/6) on the 10th day after conception. Doses of 1250, 2500 and 5000 mg/kg were given. The experiment was performed to ascertain whether the substance might have a mutagenic effect on somatic cells in vivo. The mouse spot test system permits the detection of induced point mutations and other genetic events in the melanoblasts of embryos exposed in utero. The induction of mutation is monitored post-natally by examination of the fur of young mice for recessive spots (RS) resulting from expression of recessive genes involved in coat-colour determination (Ciba Geigy 1986, Val. 2). The average litter size was not markedly affected by any of the doses administered. The survival rate of the young animals at the beginning of the observation period (approx. the 12 th day) was decreased with increasing doses. Altogether 899 animals were examined for spots. 0.29 % of the control animals showed RS. The frequencies of RS in the group treated with 1250, 2500 and 5000 mg/kg were respectively 0.93, 0 and 0 %. Thus, the relative incidence of RS among the animals treated with the various doses of the test material did not differ significantly from that of the control (sesame oil). By contrast, the positive control experiment with ethylnitrosourea (50 mg/kg) performed simultaneously yielded an statistically significant average RS frequency of 4.75 %.

In both of these in-vivo tests, also the test material TK 13143 (crude copper phthalocyanine) was used which had produced slightly positive effects in in-vitro tests (see above). However, in these in-vivo experiments no evidence for mutagenic effects were obtained.

 

CAS No. 1328-53-6:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro.

Gene mutation in bacteria

Polychloro copper phthalocyanine was not mutagenic in a preincubation Ames test (tested up to 5000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with metabolic activation, up to 625 µg/plate in S. typhimurium TA100, TA1535, TA98 and TA1537 as well as up to 5000 µg/plate in E. coli WP2 uvrA without metabolic activation; metabolic activation: Rat liver, induced with phenobarbital and 5,6-benzoflavone. Cytotoxicity was observed at 313 µg/plate or more (TA100 and TA1535) and at 625 µg/plate (TA98 and TA1537) without metabolic activation, and at 5000 µg/plate (TA100, TA1535, TA98 and TA1537) with metabolic activation (acc. Guidelines for Screening Mutagenicity Testing of Chemicals Japan, JETOC 2001).

Polychloro copper phthalocyanine was also not mutagenic in a preincubation Ames test with and without metabolic activation (tested up to 5000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with and without metabolic activation; metabolic activation: Rat liver, induced with phenobarbital and 5,6-benzoflavone. Cytotoxicity was not observed.

In another Ames test a pre-incubation assay and a standard plate test were conducted, both with and without metabolic activation (tested up to 5000μg/plate in Salmonella typhimurium strains TA100, TA98, TA1535 and TA1537; metabolic activation: S9 fraction from the liver of male Sprague-Dawley rats (treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice) and mixed with a series of cofactors. Polychloro copper phthalocyanine was also not mutagenic in this assay (comp. OECD 471, BASF AG 1988). No cytotoxicity was observed.

In another Ames test, a weakly positive result was obtained at concentrations >= 333 µg/plate in S. typhimurium TA98 only with metabolic activation (comp. OECD 471, Zeiger 1988). The mutagenicity potential of the test substance was tested up to 10000μg/plate in two Salmonella typhimurium strains TA100 and TA98; metabolic activation: S9-mix, prepared from the liver of either male SD rats or male Syrian hamsters, induced with Aroclor 1254.Without metabolic activation in the strain TA98 as well as in the strain TA100 (with or without metabolic activation), no increase of the number of his+ revertants was observed. Since the analyzed purity of the test substance used in this study was indicated to be 94 % and no further information on the remaining content was given, this result may be due to impurities of the test substance.

Another study provided also negative results for polychloro copper phthalocyanine in the Ames test (Milvy 1978, Val. 4).

Cytogenicity in mammalian cells

In a Mammalian Cell Micronucleus Test (acc. OECD 487 draft proposal, BASF AG 2001) V79 cells were used, with and without metabolic activation. The test substance was found to be negative for causing cytogenicity at dose levels from 0.78 µg/ml up to 75 µg/ml. Cytotoxicity was not observed.

CHL/IU cells were used in two in-vitro chromosomal aberration tests (acc. Japanese Guidelines for Screening Mutagenicity Testing of Chemicals, JETOC 1995 and 2001), with and without metabolic activation. The test substance was found to be negative for causing cytogenicity at dose levels up to 5000 µg/ml. Cytotoxicity was not observed.

 

CAS No. 574-93-6:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro.

Gene mutation in bacteria

Heliogen Blue MFA was not mutagenic in a preincubation in a standard plate Ames test (tested up to 5000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with and without metabolic activation (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). Cytotoxicity was not observed (acc. OECD guideline 471, BASF AG 2000).

Heliogen Blue MFA was also not mutagenic in a preincubation test with and without metabolic activation (tested up to 5000μg/plate in Salmonella typhimurium TA98 and TA100 with and without metabolic activation; metabolic activation: Rat liver, induced with phenobarbital and 5,6-benzoflavone. Cytotoxicity was not observed.

In two further tests, standard plate tests were conducted, both with and without metabolic activation, tested up to 5000μg/plate in Salmonella typhimurium strains TA100, and TA98. Polychloro copper phthalocyanine was also not mutagenic in this assays (comp. OECD 471, BASF AG 1993). No cytotoxicity was observed.

In another Ames test, a weakly positive result was obtained. Without S9-mix, no increase in the number of his+ revertants was seen in all strains tested. With S9-mix, no increase in the number of his+ revertants was seen for the strains S. typhimurium TA 100. Weakly positive reactions were seen with a maximum increase in the number of mutant colonies at 6000µg/plate for TA 98. Since the analyzed purity of the test substance used in this study was unknown, this result may be due to impurities of the test substance (comp. OECD 471, BASF AG 1993).

Another study with limited validity also provided negative results for polychloro copper phthalocyanine in the Ames test (BASF AG 1993, Val. 4).

 

CAS No. 14302-13-7:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro.

Gene mutation in bacteria

Heliogen Green 8GA was not mutagenic in a preincubation in a standard plate Ames test (tested up to 5000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with and without metabolic activation (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). Cytotoxicity was occasionally observed depending on the strain and test conditions from about 500 µg - 2500 µg/plate onward. (acc. OECD guideline 471, BASF AG 2000).

 

CAS No. 27614-71-7:

GENOTOXICITY IN-VITRO:

Valid experimental data were available to assess the genetic toxicity in-vitro.

Gene mutation in bacteria

Heliogen Blue K 6915 was not mutagenic in a preincubation and in a standard plate Ames test (tested up to 5000μg/plate in Salmonella typhimurium TA1535, TA1537, TA98, TA100 and E. coli WP2 uvrA with and without metabolic activation (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). Cytotoxicity was not observed (acc. OECD guideline 471, BASF AG 2000).

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.