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Administrative data

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:


Ames test:


- S. typhimurium TA1535, TA1537, TA98 and TA100, with and without metabolic activation (Ames test): negative (comp. OECD TG 471; BASF 1985, K2)


- S. typhimurium TA97, TA98, TA98, TA100 and TA102, with and without metabolic activation (Ames test): negative (comp. OECD TG 371, JETOC 1995, K2)


 


In vitro cytogenicity:


- CHL cells (in vitro chromosomal aberration test), with and without metabolic activation: negative (acc. Guidelines for Screening Mutagenicity Testing of Chemicals (JETOC 1995, K1).


 


DNA damage and repair:


Rat hepatocytes (DNA damage and repair): negative (OECD TG 482, Ciba Geigy 1985, K1)


Human fibroblast CRL 1521(DNA damage and repair): negative (OECD TG 482) (Ciba Geigy 1985, K1)


 


CAS No. 1328-53-6:


Ames test:


- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A, with and without met. act. (Ames test): Negative (OECD TG 471, MHLW 2000, K1)


- S. typhimurium TA1535, TA100, TA1537 and TA 98, with and without metabolic activation (Ames test): negative (comp. OECD TG 471, BASF 1988, K2)


- S. typhimurium TA 98 and TA 100, with and without metabolic activation (Ames test): weak positive effect in S. typhimurium TA98 with metabolic activation only (Zeiger 1988, K2)


 


In vitro cytogenicity:


- Chinese hamster lung fibroblasts (V79), with and without metabolic activation: Negative (MNT, OECD TG 487, BASF 2001, K1)


- CHL/IU cells, with and without metabolic activation: Negative (OECD TG 473) (Mitsubishi 2000, K2)


 


In vitro gene mutation in mammalian cells:


- Chinese hamster Ovary (CHO), with and without met. act.: Negative (HPRT, OECD TG 476, BASF 2021, K1)


 


CAS No. 574-93-6:


Ames test:


- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A (with and without metabolic activation): negative (~OECD TG 471, BASF 2000, K1)


- S. typhimurium TA 98 and TA 100: negative (~OECD TG 471, BASF 1993, K2)


- S. typhimurium TA 98 and TA 100: positive for S. typhimurium TA 98 (with metabolic activation), negative for S. typhimurium TA 100 (with and without metabolic activation) (~OECD TG 471, BASF 1993, K2)


- S. typhimurium TA 98 and TA 100: negative (~OECD TG 471, BASF 1993, K2)


- S. typhimurium TA 98 and TA 100: negative (~OECD TG 471, BASF 1993, K2)


 


CAS No. 68987-63-3:


Ames test:


- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (with and without met. act. (Ames test): Negative (OECD TG 471, Safepharm 1994, K1)


 


In vitro cytogenicity:


- Chinese hamster lung (CHL) cell line, with and without met. act.: Negative (OECD TG 473, DIC 1997, K1)


 


CAS No. 27614-71-7


Ames test:


- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (with and without met. act. (Ames test): Negative (OECD TG 471, BASF 2001, K1)


 


CAS No. 68512-13-0 


Ames test:


- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without met. act. (Ames test): Negative (OECD TG 471, BASF 2005, K1)


- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without met. act. (Ames test): Negative (OECD TG 471, BASF 2005, K1)


- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (with and without met. act. (Ames test): Negative (OECD TG 471, BASF 2005, K1)

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The study was conducted similar to OECD guideline with the following restrictions: Only 4 strains of bacteria (S. typhimurium TA102, TA97, TA100 and TA98) were tested, 5 strains are recommended in OECD guideline 471.
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 5th strain as recommended in the latest version of OECD guideline 471 (July 1997).
Principles of method if other than guideline:
The test was conducted according to the test method described by Maron and Ames (Mutat. Res. 113: 173, 1983). The rate of induced back mutations was tested in the S. typhimurium indicator organisms TA102, TA100, TA98 and TA97. However, the mutagenic potential of the test substance was not tested in an additional 5th strain, as recommended in the latest version of OECD guideline 471.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: technical grade
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:
other: S. typhimurium TA 100, TA 98, TA 102, TA 97
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction from the liver of rats, treated with KC-400 (equivalent to PCB).
Test concentrations with justification for top dose:
0, 25, 50, 100, 250, 500, 1000, 2500, 5000 µg/plate.
Vehicle / solvent:
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:
- A preincubation assay was conducted according to the test method described by Maron and Ames (Mutat. Res. 113: 173, 1983).

- Positive controls:
without S9-mix: AF-2(TA 100, TA98), Mitomycin C (TA 102) and ICR-191 (TA97);
with S9-mix: 2-Aminoanthracene (all strains) S-9: rat liver, induced with KC-400 (equivalent to PCB)

- Plates per dose: 2

- Number of replicate: 1
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, other: TA 100, TA 98, TA 102, TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
no bacteriotoxic effect was observed at 5000 µg/plate with or without S9-mix.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Cytotoxicity: No toxicity was observed at a concentration of 5000 µg/plate with or without metabolic activation.

- 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).
The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

Table 1: Revertants/plate in the preincubation test

Compound

Solvent

Concentration (µg/plate)

TA100

TA98

TA102

TA97

 

 

 

without S9

with S9

without S9

with S9

without S9

with S9

without S9

with S9

test material

DMSO

0

175

147

16

33

335

477

198

294

 

 

25

133

154

20

33

360

559

286

313

 

 

50

160

161

30

28

376

532

221

325

 

 

100

226

151

17

35

385

574

189

302

 

 

250

174

171

24

26

382

627

192

258

 

 

500

177

153

24

25

426

456

215

267

 

 

1000

156

124

21

30

361

383

195

239

 

 

2500

114

123

29

32

350

375

201

274

 

 

5000

153

133

19

27

321

410

177

257

AF2 (TA 100)

DMSO

0.02

1010

 

 

 

 

 

 

 

AF2 (TA 98)

DMSO

0.05

 

 

207

 

 

 

 

 

MMC

distilled water

0.01

 

 

 

 

1386

 

 

 

ICR-191

DMSO

0.1

 

 

 

 

 

 

840

 

2-AA (TA100, TA98, TA97)

DMSO

2

 

2982

 

2698

 

 

 

1400

2-AA (TA102)

DMSO

10

 

 

 

 

 

2504

 

 

AF2 = Furylfuramide

MMC = Mitomycin C

ICR-191 = 6-Chloro-9-(3-[2-chloroethylamino]propylamino)-2-methoxyacridine] dihydrochloride

2-AA = 2-Aminoanthracene

Conclusions:
An increase in the number of his+ revertants was not seen in the preincubation test (with or without metabolic activation).
According to the results of the present study, the tested substance was not mutagenic in the Ames test under the experimental conditions chosen here.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Peer reviewed study with acceptable restrictions.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; only two S. typhimurium strains (TA98 and TA100) were used.
Principles of method if other than guideline:
A preincubation assay was conducted according to the method described by Haworth et al. (Environ Mutagen 5, Suppl. 1: 3-142). Only two S. typhimurium strains (TA98 and TA100) were used for evaluation of the mutagenic potential of the test material.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 94 %
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:
other: Salmonella typhimurium TA98 and TA100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix, prepared from the liver of either male SD rats or male Syrian hamsters, induced with Aroclor 1254.
Test concentrations with justification for top dose:
0, 100, 333, 1000, 3333, 6666, 10000 µg/ plate
Vehicle / solvent:
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 and conditions"
Details on test system and experimental conditions:
The test material was tested initially in a toxicity assay using the Salmonella strain TA100 to determine the appropriate dose range.
Mutagenicity testing was conducted in strains S. typhimurium TA98 and TA100. The test chemical (0.05 ml), Salmonella typhimurium culture (0.10 ml), and S-9 mix or buffer alone (0.50 ml) were incubated at 37 °C, without shaking, for 20 min. The top agar was added and the contents of the tubes
were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium. The his+ colonies arising on the plates were machine-counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the colour of the test chemical on the plate reduced the contrast between the colonies and the background agar. At least 5 doses of each chemical were tested in triplicates. Plates with low numbers of colonies were counted by hand. Testing wasconducted without metabolic activation and with 30 % rat-, or 30 % hamster S9-mix. If the result obtained was positive in at least one strain, the test was repeated. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate.

Controls:
Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA100) and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation used for both strains was 2-aminoanthracene.
Evaluation criteria:
Evaluations were made at both the individual trial and overall chemical levels.
Individual trials were judged mutagenic (+), weakly mutagenic (+W), questionable or nonmutagenic (-), depending on the magnitude of die increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of his+ if only a single dose was elevated over the control, or if the inerease seen was not dose related. The distinctions between a questionable mutagenic response and a nonmutagenic or weak mutagenic response, and between a weak mutagenic response and mutagenic response are highly subjective. lt was not necessary for a response to reach twofold over background for a chemical to be judged mutagenic.
A chemical was judged mutagenic (+) or weakly mutagenic (+W) if it produced a reproducible dose-related reponse over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged nonmutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity.

Species / strain:
S. typhimurium, other: strains TA98 and TA100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
; weak positive effect in S. typhimurium TA98 with metabolic activation only.
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
When tested in TA 100, no concentration related increase of revertants was observed irrespective of the presence or absence of metabolic activation. Using TA 98, a slight increase of revertants was seen at concentrations >= 333 µg/plate in a dose-dependent manner only in the presence of metabolic activation (for both rat S9-mix and hamster S9-mix). Those minor increases where all noted at concentrations, at which precipitation of the test substance was concurrently observed.
Negative as well as positive controls showed appropriate responses.

Table 1: Number of revertants per plate in S. typhimurium TA98 and TA100

Dose (µg/plate)

TA 100

 

 

TA 98

 

 

 

NA

30 % HLI

30 % RLI

NA

30 % HLI

30 % RLI

0

134 ± 6.0

100 ± 11.9

138 ± 5.0

17 ± 1.2

30 ± 1.8

30 ± 1.2

100

122 ± 4.7

96 ± 6.5

131 ± 9.1

20 ± 1.5

27 ± 2.5

 -

333

93 ± 5.9 p

109 ± 11.4 p

138 ± 14.7 p

14 ± 2.9 p

36 ± 3.2 p

36 ± 5.2 p

1000

103 ± 12.5 p

118 ± 7.8 p

151 ± 6.2 p

18 ± 3.2 p

59 ± 3.5 p

52 ± 5.2 p

3333

107 ± 8.3 p

139 ± 8.1

145 ± 6.6 p

20 ± 5.4 p

93 ± 6.9 p

73 ± 3.9 p

6666

 -

 -

- 

- 

 -

94 ± 3.2 p

10000

108 ± 9.5 p

140 ± 12.6 p

157 ± 9.0 p

14 ± 0.7 p

130 ± 10.0 p

87 ± 5.0 p

pos. Control

281 ± 8.0

397 ± 9.8

413 ± 6.8

406 ± 1.5

309 ± 19.1

162 ± 7.7 p

 
NA = not activated, no S9-mix
HLI = Aroclor 1254-induced hamster liver S9-mix
RLI = Aroclor 1254-induced rat liver S9-mix
p = precipitate present in plates
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997 (required only 4 strains)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 98 %
- Batch No.: BW 22
- Storage condition of test material: 4 °C
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:
other: Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98
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:
1st experiment: Standard plate test
0, 20, 100, 500 and 5000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537), with or without S9-mix.

2nd Experiment: Preincubation test
0, 20, 100, 500, 2500 and 5000 µg/plate (tested in the strains S. typhimurium T98, TA100, TA1535 and TA1537), with or without S9-mix.
Vehicle / solvent:
- Vehicle/solvent 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 test and a preincubation test were conducted, both with and without metabolic activation (S9 mix). Each test was conducted in triplicates. The test system is inadequate for evaluating cross-linking mutagens.

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.

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, 0.1 ml bacterial suspension and 0.5 ml S-9 mix are incubated at 37°C for the duration of 20 minutes. Subsequently, 2 ml of soft agar is added and, after mixing, the samples are poured onto the Vogel-Bonner agar plates within approx. 30 seconds.
Composition of the minimal glucose agar:
980 ml aqua dest.
20 ml Vogel-Bonner E medium
15 g Difco bacto agar
20 g D-glucose monohydrate.
After incubation at 37 °C for 48 hours in the dark, the bacterial colonies (his+ revertants) are counted.

CONTROLS:
- Negative control:
Parallel with each experiment with and without S-9 mix, a negative control (with vehicle 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 amounts of 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.


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, other: Salmonella typhimurium strains TA1535, TA100, TA1537 and TA98
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:
SOLUBILITY: Incomplete solubility of the test substance in DMSO was seen from about 500 µl/plate onward.

TOXICITY: No bacteriotoxic effect (reduced his- background growth) was observed

CONTROLS: Negative as well as positive controls showed appropriate responses.

MUTAGENICITY: An increase in the number of his+ was not observed both 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

16 ± 3

20 ± 3

Test substance

19 ± 5 [100]

21 ± 3 [500]

Positive Control

1693 ± 169 [5; MNNG]

231 ± 13 [10; 2 -AA]

S. typhimurium TA100

DMSO

114 ± 5

116 ± 7

Test substance

128 ± 4 [2500]

122 ± 3 [500]

Positive Control

2117 ± 29 [5; MNNG]

2050 ± 0 [10; 2 -AA]

S. typhimurium TA1537

DMSO

9 ± 2

9 ± 1

Test substance

12 ± 3 [100]

12 ± 1 [500]

Positive Control

777 ± 17 [10; NOPD]

156 ± 23 [10; 2 -AA]

S. typhimurium TA98

DMSO

21 ± 1

35 ± 4

Test substance

22 ± 1 [2500]

35 ± 4 [100]

Positive Control

871 ± 22 [100; AAC]

1513 ± 32 [10; 2 -AA]

2-AA = 2-aminoanthracene  

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

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

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

16 ± 2

19 ± 2

Test substance

21 ± 4 [500]

19 ± 2 [5000]

Positive Control

629 ± 8 [5; MNNG]

149 ± 20 [10; 2 -AA]

S. typhimurium TA100

DMSO

115 ± 5

114 ± 11

Test substance

127 ± 7 [2500]

126 ± 11 [20]

Positive Control

1540 ± 87 [5; MNNG]

2200 ± 50 [10; 2 -AA]

S. typhimurium TA1537

DMSO

8 ± 1

8 ± 1

Test substance

9 ± 2 [2500]

10 ± 3 [500]

Positive Control

883 ± 98 [10; NOPD]

186 ± 23 [10; 2 -AA]

S. typhimurium TA98

DMSO

22 ± 1

32 ± 2

Test substance

24 ± 5 [500]

36 ± 7 [500]

Positive Control

725 ± 59 [100; AAC]

1317 ± 126 [10; 2 -AA]

2-AA = 2-aminoanthracene  

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

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      
Conclusions:
non mutagenic in bacteria
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; the mutagenic potential of the test material was investigated in a reduced Ames test screening programme; only the two S. typhimurium strains TA98 and TA 100 were tested.
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 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
1st experiment:
Standard plate test with and without S9-mix ( in S. typhimurium TA98 and TA100): 0, 100, 500, 2500 and 5000 µg per plate
2nd experiment:
Standard plate test with and without S9-mix (in S. typhimurium TA98): 0, 2000, 4000, 6000 and 8000 µg per plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
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:
A standard plate test was conducted, with and without metabolic activation (S9 mix) in the S. typhimurium strains TA98 and TA100. Each test was conducted in triplicates.

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.

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 Salmonella strains); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98).
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed.

MUTAGENICITY:
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 6000 µg/plate (factor 2.9-3.0) for TA 98.

SOLUBILITY:
Incomplete solubility of the test substance in solvent DMSO was seen from about 500 µg/plate onward.

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 TA100

DMSO

101 ± 17

100 ± 2

Test substance

93 ± 9 [2500]

94 ± 9 [500]

Positive Control

1343 ± 101 [5; MNNG]

894 ± 35 [2.5; 2-AA]

S. typhimurium TA98

DMSO

25 ± 1 

36 ± 4

Test substance

25 ± 3 [4000]

104 ± 10 [6000]

Positive Control

751 ± 51 [10; NOPD]

667 ± 20 [2.5; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  
Conclusions:
According to the results of the present study, the test substance was weakly mutagenic in the Ames test in the strain S. typhimurium TA98 with metabolic activation under the experimental conditions chosen here. However, the very slight increase in the number of revertant colonies was only observed at high doses at which the dyestuff precipitated on the agar plates; therefore, impurities rather than the test substance itself might be the cause for these findings.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; the mutagenic potential of the test material was investigated in a reduced Ames test screening programme; only the two S. typhimurium strains TA98 and TA 100 were tested.
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 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
First experiment:
100, 500, 2500 and 5000 ug/plate (TA 100)
100, 500, 2500 and 5000 ug/plate (TA 98)
Second experiment:
2000, 4000, 6000 and 8000 µg/plate (TA98)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
other: With S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine
Details on test system and experimental conditions:
A standard plate test was conducted, with and without metabolic activation (S9 mix) in the S. typhimurium strains TA98 and TA100. Each test was conducted in triplicates.

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.

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 Salmonella strains); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98).
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed.

MUTAGENICITY:
No increase in the number of his+ revertants was seen in all strains tested, with or without S9-mix.

SOLUBILITY:
Incomplete solubility of the test substance in solvent DMSO was seen from about 500 µg/plate onward.

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 TA100

DMSO

101 ± 17

100 ± 2

Test substance

95 ± 4 [5000]

93 ± 5 [500]

Positive Control

1343 ± 101 [5; MNNG]

894 ± 35 [2.5; 2-AA]

S. typhimurium TA98

DMSO

25 ± 1  

36 ± 4

Test substance

30 ± 2 [500]

41 ± 1 [5000]

Positive Control

751± 51 [10; NOPD]

667 ± 20 [2.5; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  
Conclusions:
According to the results of the present study, the test substance was not mutagenic in the Ames test in the strains S. typhimurium TA98 or TA100 with or without metabolic activation under the experimental conditions chosen here.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; the mutagenic potential of the test material was investigated in a reduced Ames test screening programme; only the two S. typhimurium strains TA98 and TA 100 were tested.
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 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
Standard plate test with and without S9-mix ( in S. typhimurium TA98 and TA100): 0, 100, 500, 2500 and 5000 µg per plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
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:
A standard plate test was conducted, with and without metabolic activation (S9 mix) in the S. typhimurium strains TA98 and TA100. Each test was conducted in triplicates.

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.

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 Salmonella strains); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98).
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed.

MUTAGENICITY:
No increase in the number of his+ revertants was seen in all strains tested, with or without S9-mix.

SOLUBILITY:
Incomplete solubility of the test substance in solvent DMSO was seen from about 2500 µg/plate onward.

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 TA100

DMSO

103 ± 14

104 ± 10

Test substance

120 ± 3 [100]

108 ± 12 [100]

Positive Control

1020 ± 184 [5; MNNG]

637 ± 95 [2.5; 2-AA]

S. typhimurium TA98

DMSO

31 ± 3 

41 ± 2

Test substance

35 ± 1 [500]

41 ± 5 [500]

Positive Control

1258 ± 111 [10; NOPD]

711 ± 36 [2.5; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  
Conclusions:
Weakly positive under the test conditions chosen. The positive reaction was seen only in high concentrations and it cannot be excluded, that impurities were the cause for the positive reaction.
According to the results of the present study, the test substance was not mutagenic in the Ames test in the strains S. typhimurium TA98 or TA100 with or without metabolic activation under the experimental conditions chosen here.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
other information
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
; the mutagenic potential of the test material was investigated in a reduced Ames test screening programme; only the two S. typhimurium strains TA98 and TA 100 were tested.
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 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
rat liver S-9 mix
Test concentrations with justification for top dose:
Standard plate test with and without S9-mix ( in S. typhimurium TA98 and TA100): 0, 100, 500, 2500 and 5000 µg per plate
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
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:
A standard plate test was conducted, with and without metabolic activation (S9 mix) in the S. typhimurium strains TA98 and TA100. Each test was conducted in triplicates.

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.

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 Salmonella strains); without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, MNNG (5 µg/plate for TA100); 4-nitro-o-phenylenediamine, NOPD (10 µg/plate for TA 98).
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
No bacteriotoxic effect (reduced his- background growth) was observed.

MUTAGENICITY:
No increase in the number of his+ revertants was seen in all strains tested, with or without S9-mix.

SOLUBILITY:
Incomplete solubility of the test substance in solvent DMSO was seen from about 2500 µg/plate onward.

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 TA100

DMSO

103 ± 14

104 ± 10

Test substance

109 ± 11 [100]

121 ± 14 [100]

Positive Control

1020 ± 184 [5; MNNG]

637 ± 95 [2.5; 2-AA]

S. typhimurium TA98

DMSO

31 ± 3

41 ± 2

Test substance

37 ± 3 [500]

46 ± 7 [500]

Positive Control

1258 ± 111 [10; NOPD]

711 ± 36 [2.5; 2-AA]

2-AA = 2-aminoanthracene  

MNNG = N-methyl-N'-nitro-N-nitrosoguanidine  
Conclusions:
According to the results of the present study, the test substance was not mutagenic in the Ames test in the strains S. typhimurium TA98 or TA100 with or without metabolic activation under the experimental conditions chosen here.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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:
mammalian cell line, other: Chinese hamster lung (CHL) cell line
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:
mammalian cell line, other: CHL (Chinese hamster lung) cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: none
- Water solubility: substance is insoluble
- Precipitation: substance is applied as a suspension
- Other confounding effects:

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

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

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

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

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

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

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

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

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

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

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

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

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
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
Specific details on test material used for the study:
-Test item name: Blue 10 GN
- Other name in report: Copper, [29H, 31H-phthalocyaninato-(2-)-N29, N30 , N31 , N32]-chlorinated (n=8-13)
- CAS 68987-63-3
-Batch no. 21993
-Purity: 99%
-Description: blue powder
-Storage conditions: Room temperature
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
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
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.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

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:
non mutagenic in the Ames test
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot 029008P050
Green powder
Storage Room temperature
Year of manufacture: 2003
Target gene:
his operon
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:
S9 mix from liver of Aroclor 1254-induced rats
Test concentrations with justification for top dose:
0; 20; 100; 500; 2500 and 5000 microgramm/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
other: 2-Aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation) for the first experiment and preincubation for the repeat experiment
Evaluation 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
S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
*The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in 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 was within the normal range of
the historical control data for each tester strain (see Appendix 5).
" The sterility controls revealed no indication of bacterial contamination.
" The positive control articles both with and without S-9 mix induced a significant increase in
the number of revertant colonies within the range of the historical control data or above
(see Appendix 6).
" The titer of viable bacteria was > 108/mI.
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
E. coli WP2 uvr A
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 bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed.
Test substance precipitation was found from about 100 microgramm/plate onward.
The test substance did not Iead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

E.coli. WP2 uvrA without S9 with S9
Dose/Plate Revertants Number SD Factor Revertants Mean SD Factor Titre
DMSO 28 31 3 1.0 30 34 5 1.0 26
31 39 30
34 34 30
20 ug 30 34 4 1.1 31 34 3 1.0
34 36
37 35
100 ug 30P
27P		 32 7 1.0 35P
33P		 32 4 0.9
40P 27P
500 ug 28P
29P		 29 1 0.9 33P
36P		 35 2 1.0
30P 36P
2500 ug 29P 29 2 0.9 32P 32 3 0.9 21
27P 35P 33
30P 30P 25
5000 ug 28P 26 5 0.8 36P 32 3 0.9 27
29P 31P 30
20P 30P 25
4-NQO 5.0 ug 705 673 28 21.7
661
653
2-AA 60 ug 250 226 22 6
207
222

Tables from the plate incorporation test are provided in the attachment.

Conclusions:
The substance is not mutagenic in the Ames test.
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot 020036P050
Green powder
Storage Room temperature
Year of manufacture: 2003
Target gene:
his operon
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:
S9 mix from liver of Aroclor 1254-induced rats
Test concentrations with justification for top dose:
0; 20; 100; 500; 2500 and 5000 microgramm/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
other: 2-Aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation) for the first experiment and preincubation for the repeat experiment
Evaluation 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
S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
*The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in 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 was within the normal range of
the historical control data for each tester strain (see Appendix 5).
" The sterility controls revealed no indication of bacterial contamination.
" The positive control articles both with and without S-9 mix induced a significant increase in
the number of revertant colonies within the range of the historical control data or above
(see Appendix 6).
" The titer of viable bacteria was > 108/mI.
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
E. coli WP2 uvr A
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 bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed.
Test substance precipitation was found from about 100 microgramm/plate onward.
The test substance did not Iead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Tables with results of the plate incorporation and preincubation assay are provided in the attachment.

Conclusions:
The substance is not mutagenic in the Ames test.
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Lot 020032P050
Green powder
Storage Room temperature
Year of manufacture: 2003
Target gene:
his operon
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:
S9 mix from liver of Aroclor 1254-induced rats
Test concentrations with justification for top dose:
0; 20; 100; 500; 2500 and 5000 microgramm/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
other: 2-Aminoanthracene, N-methyl-N`-nitro-N-nitrosoguanidine, 4-nitro-phenylendiamine
Details on test system and experimental conditions:
METHOD OF APPLICATION:in agar (plate incorporation) for the first experiment and preincubation for the repeat experiment
Evaluation 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
S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
*The number of revertants for all tester strains were within the historical negative control
range under all experimental conditions in 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 was within the normal range of
the historical control data for each tester strain (see Appendix 5).
" The sterility controls revealed no indication of bacterial contamination.
" The positive control articles both with and without S-9 mix induced a significant increase in
the number of revertant colonies within the range of the historical control data or above
(see Appendix 6).
" The titer of viable bacteria was > 108/mI.
Species / strain:
S. typhimurium TA 1535
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
Species / strain:
S. typhimurium TA 1537
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
Species / strain:
S. typhimurium TA 98
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
Species / strain:
S. typhimurium TA 100
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
Species / strain:
E. coli WP2 uvr A
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 bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed.
Test substance precipitation was found from about 100 microgramm/plate onward.
The test substance did not Iead to an increase in the number of revertant colonies either without S-9 mix or after adding a metabolizing system in two experiments carried out independently of each other (standard plate test and preincubation assay).

Tables are provided in the attachment.

Conclusions:
The substance is not mutagenic in the Ames test.
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:
- Analytical purity: 97.42 %
- 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 (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:
other: With S-9 mix: 2-aminoanthracene; without S-9 mix: N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine and 4-nitroquinoline-N-oxide.
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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
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
True negative controls validity:
not applicable
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:
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.
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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not applicable
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TOXICITY:
No bacteriotoxic effect was observed.

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

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

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

    

Strain

Tested compound

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

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

21 ± 2

23 ± 2

Test substance

23 ± 4 [2500]

25 ± 2 [100]

Positive Control

639 ± 15 [5; MNNG]

136 ± 33 [2.5; 2-AA]

S. typhimurium TA100

DMSO

108 ± 6 

109 ± 3

Test substance

104 ± 4 [20]

112 ± 5 [500]

Positive Control

887 ± 73 [5; MNNG]

809 ± 59 [2.5; 2-AA]

S. typhimurium TA98

DMSO

33 ± 2 

44 ± 4

Test substance

34 ± 5 [100]

57 ± 8 [5000]

Positive Control

712 ± 33 [10; NOPD]

572 ± 46 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

10 ± 2

10 ± 3

Test substance

9 ± 3 [20]

12 ± 4 [2500]

Positive Control

609 ± 100 [100; AAC]

95 ± 3 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

48 ± 7

45 ± 3

Test substance

45 ± 13 [20]

52 ± 11 [500]

Positive Control

734 ± 46 [5; 4-NQO]

228 ± 26 [60; 2-AA]

2-AA = 2-aminoanthracene  

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

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      

 

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

    

Strain

Tested compound

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

 

 

without S9-mix

with S9-mix

S. typhimurium TA1535

DMSO

17 ± 2

17 ± 1

Test substance

17 ± 2 [100]

16 ± 2 [20]

Positive Control

795 ± 40 [5; MNNG]

203 ± 6 [2.5; 2-AA]

S. typhimurium TA100

DMSO

106 ± 6

109 ± 7

Test substance

108 ± 6 [20]

107 ± 4 [20]

Positive Control

1094 ± 105 [5; MNNG]

889 ± 68 [2.5; 2-AA]

S. typhimurium TA98

DMSO

28 ± 5

31 ± 3

Test substance

30 ± 7 [20]

41 ± 4 [5000]

Positive Control

877 ± 68 [10; NOPD]

632 ± 63 [2.5; 2-AA]

S. typhimurium TA1537

DMSO

9 ± 2

9 ± 2

Test substance

9 ± 3 [100]

9 ± 1 [20]

Positive Control

436 ± 18 [100; AAC]

94 ± 5 [2.5; 2-AA]

E. coli WP2 uvrA

DMSO

38 ± 5

29 ± 5

Test substance

39 ± 4 [5000]

35 ± 6 [2500]

Positive Control

483 ± 102 [5; 4-NQO]

223 ± 7 [60; 2-AA]

2-AA = 2-aminoanthracene  

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

NOPD = 4-nitro-o-phenylenediamine      

AAC = 9-aminoacridine      

4-NQO = 4-nitroquinoline-N-oxide      
Conclusions:
According to the results of the present study, the test substance did not lead to an increase in the number of his revertant colonies either with or without S9-mix in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions chosen here.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
A positive control (benzo(a)pyrene) was used, however, the results of the positive control were not listed.
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Analytical purity: technical grade
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:
other: Chinese Hamster CHL cells
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:
mammalian cell line, other: Chinese Hamster CHL cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
An increase in the number of chromosomal aberrations was not seen in the Chromosomal Aberration test, neither after continuous treatment with the test substance for 24 or 48 h without S9-mix, nor after pulse treatment for 6 h followed by harvesting at 24 h, with or without metabolic activation.
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.
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:
comparable to 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:
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 assay, 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, 12, 2.40 and 0.48 µg/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

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.

Conclusions:
negative without metabolic activation

The test substance did not induce DNA-repair activiity in a human fibroblast cell line that was exposed for 5h without additional metabolic activation.
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:
comparable to 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 assay, 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, 12, 2.40 and 0.48 µg/ml

DNA-repair test (repeat experiment):
0.1, 0.2, 0.4, 0.6, 1, 2, 4, 6, 10, 20, 40 and 60 µg/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:
not applicable
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

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

Treatment group concentration Silver grain per nucleus-equivalent area ± SD
control (medium) n.a. 1,47 ± 1,16
control (Vehicle) n.a. 1.29 ± 1.08
positive control DMN 100 mM 20.1 + 6.89
Test substance  60.0 µg/ml 2.06 ± 1,53
Test substance 12.0 µg/ml 1.87 + 1.11
Test substance 2.4 µg/ml 1.76 ± 1.36
Test substance 0.48 µg/ml 2.27 ± 1.37
Conclusions:
negative
Endpoint:
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:
other: Well documented study with the following restriction: Only 4 strains of bacteria (S. typhimurium TA1535, TA1537, TA100 and TA98) were tested, 5 strains are recommended in OECD guideline 471 (July 1997). No E. coli strain was tested.
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
Specific details on test material used for the study:
- Analytical purity: ca. 98 %
- Impurities (identity and concentrations): no data given
- Storage condition of test material: 4 °C
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 test 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.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the standard plate test
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the standard plate test
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the standard plate test
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the standard plate test
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 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:
No increase in the number of his+ revertants was seen in the standard plate test (with or without metabolic activation) in all tested S. typhimurium strains (TA1535, TA100, TA1537, TA98).
According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Analytical purity: 99.1 %
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:
other: CHL/IU cells
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:
other: -S9-mix: Mitomycin C; +S9-mix: Benzo[a]pyrene
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:
mammalian cell line, other: Chinese hamster CHL/IU cells
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
-------------------------------------------------------------------------- ------------

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)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Analytical purity: 99.1 %
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
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
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
True negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium 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
True negative controls validity:
not applicable
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:
The test substance is not mutagenic in the Ames test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25.1.2021 - June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
29 Jul 2016
Deviations:
yes
Remarks:
Additional investigations for nanomaterials included
GLP compliance:
yes (incl. QA statement)
Remarks:
certified by Landesamt für Umwelt Rheinland-Pfalz
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 11 Feb 2022
- Purity: 99.6%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under storage conditions: stable


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test substance preparation was performed in accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June, 2018.
The test substance was weighed, pre-wetted with 0.5 vol% ethanol (pre-wetting is introduced to enable dispersion of hydrophobic materials in water-based systems) and topped up with the vehicle 0.05% w/v BSA-water to achieve the required concentration of the stock dispersions. Two stock dispersions were prepared (xx mg/mL and xxmg/mL).
The stock dispersion of xx mg/mL was handled separately. For further dilutions only the stock dispersion of xx mg/mL were used.
A homogeneous test substance preparation in the vehicle was prepared by using a Branson Sonifier S-550D (Branson Ultrasonics Corp., Danbury, CT, USA) equipped with a standard 13 mm disruptor horn.
The test substance formulations was diluted according to the planned doses.
All test substance formulations were prepared immediately before administration.
To keep the test substance homogeneously in the vehicle, the test substance preparation was carefully pipetted before the removal.


FORM AS APPLIED IN THE TEST: Homogeneous dispersion

OTHER SPECIFICS
- physical state, appearance: Solid, green
- molecular weight: 1057 - 1127 g/mol (UVCB)
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type of cells: CHO (Chinese hamster ovary) cell line
- Source: cell stock of testing facility
- Suitability of cells: yes, as recommended in OECD TG 476
- Normal cell cycle time (negative control): not specified

For cell lines:
- Absence of Mycoplasma contamination: yes
- Number of passages if applicable: 3
- Methods for maintenance in cell culture: Cell medium was removed and cells were washed with 5 mL PBS or HBSS (both Ca-Mgfree). Cells were trypsinized with 2 mL HBSS (Hanks balanced salt solution; Ca-Mg-free) and 2 mL trypsin (0.25% [w/v]) to remove the cells from the bottom of the plastic flasks. This reaction was stopped by adding 6 mL culture medium incl. 10% (v/v) FCS. Cells were pipetted up and down to separate them and to prepare a homogeneous single cell suspension. Cells were counted in a counting chamber or using a cell counter. Cell suspensions were diluted with complete culture medium to the desired cell count.
- Doubling time: of about 12 - 16 hours
- Modal number of chromosomes: 20
- Periodically checked for karyotype stability: not specified
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Ham's F12 medium containing stable glutamine and hypoxanthine (PAN Biotech; Cat. No. P04-15500) supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL); 5% (v/v) CO2 at 37°C and ≥ 90% relative humidity
Metabolic activation:
with and without
Metabolic activation system:
- type and composition of metabolic activation system: exogenous metabolic activation by cofactor-supplemented postmitochondrial fraction (S9 mix)
- source of S9: liver S9 mix from phenobarbital- and β-naphthoflavone induced rats
- method of preparation of S9 mix: the S9 mix was prepared freshly prior to each experiment. 1 part S9 fraction was mixed with 9 parts S9 supplement (consisting of 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 15 mM phosphate buffer (pH 7.4))
- concentration or volume of S9 mix and S9 in the final culture medium: 8 mL S9 mix and 32 mL test substance preparation (final volume 40 mL)
Test concentrations with justification for top dose:
1st Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL

2nd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.0)0 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; (2000.00) µg/mL

3rd Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL
with S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL

4th Experiment
without S9 mix
0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL

Doses in brackets lead to inhomogeneous suspensions and were not investigated. In lower doses, the suspensions were homogenous.
Vehicle / solvent:
- Vehicle used: 0.05% w/v bovine serum albumin water (BSA-water)
- Justification for choice of solvent/vehicle: In accordance to the “SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018, 0.05% w/v bovine serum albumin water (BSA-water) is used as vehicle.
The final concentration of the vehicle 0.05% w/v BSA-water in culture medium is be 10% (v/v).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Remarks:
- DMBA with metabolic activation (1.25 μg/mL) - EMS without metabolic activation (400 μg/mL)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 4

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 20x10^6 cells in 40 mL medium/flask


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20-24 hours
- Exposure duration/duration of treatment: 4 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 7-9 days
- Selection time: 6-7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7-9 days (cloning efficiency 1); 16 days (cloning efficiency 2)
- Method used: colonies were fixed with methanol and stained with Giemsa
- Selective agent: 6-thioguanine; 10 μg/mL; 6-7 days exposure
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2x10^6 cells from every treatment group were seeded in 20 mL selection medium (175 cm^2 flasks) and the remaining colonies were counted at the end of the selection period
- Criteria for small (slow growing) and large (fast growing) colonies: not applicable

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cloning efficiency

METHODS FOR MEASUREMENTS OF GENOTOXICITY
- Mutant frequency

- OTHER: Check or determination of further parameters:
- pH, osmolality, solubility, cell morphology
-Analytical Ultracentrifugation (AUC) method to determine the agglomeration in genotoxicity test medium.
- Incubation and filtration with analysis by Inductively-Coupled-Plasma Mass Spectrometry (ICPMS) or by UVVis-Spectrometry, to determine the static solubility in genotoxicity test medium.


Rationale for test conditions:
In the pre-test for toxicity based on the SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018, 2.56 mg/mL 29H,31H-phthalocyanine was used as stock dispersion. The highest tested concentration was 100.0 μg/mL both with and without S9 mix at 4 hour exposure time.
The pre-test was performed following the method described for the main experiment. The Relative Survival (RS) was determined as a toxicity indicator for dose selection.
In the pre-test for dose selection the pH, osmolality and solubility were additionally determined for all or at least some selected doses.
Evaluation criteria:
A test substance is considered to be clearly positive if all following criteria are met:
- A statistically significant increase in mutant frequencies is obtained.
- A dose-related increase in mutant frequencies is observed.
- The corrected mutation frequencies (MFcorr.) exceeds both the concurrent negative control value and the range of our laboratory’s historical negative control data (95% control limit).

Isolated increases of mutant frequencies above our historical negative control range or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.

A test substance is considered to be clearly negative if the following criteria are met:
- Neither a statistically significant nor dose-related increase in the corrected mutation frequencies is observed under any experimental condition.
- The corrected mutation frequencies in all treated test groups is close to the concurrent vehicle control value and within the range of our laboratory’s historical negative control data (95% control limit).
Statistics:
A linear dose-response is evaluated by testing for linear trend. The dependent variable is the corrected mutant frequency and the independent variable is the dose. The calculation is performed using EXCEL function RGP. The used model is one of the proposed models of the International Workshop on genotoxicity Test procedures Workgroup Report.

A pair-wise comparison of each test group with the control group is carried out using Fisher's exact test with Bonferroni-Holm correction. The calculation is performed using EXCEL function HYPGEOM.VERT.

If the results of these tests were statistically significant compared with the respective vehicle control, labels (s p ≤ 0.05) are printed in the tables. However, both, biological and statistical significance are considered together.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: All doses contain insoluble particles, homogenicity of suspension as criterium
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
The summary table is attached as a figure.

 

 

 

 

Conclusions:
Pigment Green 7 is not mutagenic in the HPRT test.
Executive summary:

The substance Polychloro copper phthalocyanine was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and beta-naphthoflavone induced rats (exogenous metabolic activation). Since the test substance is a nanoparticle, dose selection for genotoxicity testing was based on the SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018. Furthermore, to fulfill the requirements of the OECD Guidelines for the HPRT assay, the top concentrations in all main Experiments were defined as the highest homogenous suspension. 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle. Test groups printed in bold type were evaluated for gene mutations, except the highest tested concentration of the 1st and 2nd Experiment. 2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid. In the 3rd and 4th Experiment 256.00 µg/mL was used as top concentration which was the highest homogeneous suspension which could be applied to the test culture.

Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week.

Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In all experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in four experiments performed independently of each other.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
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
Specific details on test material used for the study:
- Batch No: BW 137 (Partien 616-619)
- Analytical purity: > 98 % (analytical report dated Feb 15, 2000)
- Appearance, consistency: green powder
- Storage: room temperature
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 of the 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:
no
Positive controls:
yes
Positive control substance:
other: Ethyl-Methane-Sulfonate (EMS) without S9-mix and Cyclophosphamide (CPP) with S-9 mix
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
----------------------------------------------------------

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 (Ciba Geigy 1986, K2)


- Gene mutation in-vivo, mouse (Mouse Spot Test): negative (Ciba Geigy 1986, K2)

Link to relevant study records

Referenceopen allclose all

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:
test procedure in accordance with national standard methods with acceptable restrictions
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:
- 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 lasted 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 of 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. A 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 variability without dose-dependency.
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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:
- 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
Conclusions:
Under the conditions of the experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test material.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

All substances in this category share a similar structure with a molecular weight > 500 g/mol. Based on the high molecular weight and insolubility, the substances in this category will not be able to penetrate biological membranes. Thus, no systemic exposure is expected. This is confirmed by the genetic toxicity studies for six substances in this category: CAS 147-14-8, CAS 1328-53-6, CAS 574-93-6, CAS 68987-63-3, CAS 27614-71-7 and CAS 68512-13-0. The genetic toxicity data can be used for read-across to all substances in this category.


 


CAS No. 147-14-8:


GENOTOXICITY IN-VITRO


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


 


Ames test:


No increase in the number of his+ revertants was seen in the standard plate test (with or without metabolic activation) in all tested S. typhimurium strains (TA1535, TA100, TA1537, TA98).
According to the results of the present study, the test substance is not mutagenic in the Ames test under the experimental conditions chosen here (BASF 1985, K2).


 


In another Ames test a pre-incubation assay was conducted with and without metabolic activation (tested up to 5000 μg/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, K2). No cytotoxicity was observed.


 


In vitro cytogenicity:


CHL cells were used in an in-vitro chromosomal aberration test (acc. Japanese Guidelines for Screening Mutagenicity Testing of Chemicals, JETOC 1995, K1), 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.


 


DNA damage and repair:


Unscheduled DNA synthesis was examined in primary rat hepatocytes and in a human fibroblast cell line. Each experiment was reported separately. Primary rat hepatocytes are metabolically competent whereas permanent cell lines such as the human fibroblasts have generally very little metabolic capacity. Both experiments did not show an induction of DNA repair activity.


In the study with human fibroblasts (Ciba Geigy 1985, K1), 60.0, 12.0, 2.40 and 0.48 µg/mL were 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 visualised 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 (5 µM) 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.


In the study with primary rat hepatocytes (Ciba Geigy 1985, K1), eleven concentrations increasing from 0.62 µg/mL to 100 µg/mL were tested to determine the highest applicable concentration in the DNA-repair assay. At the three highest concentrations (60, 80 and 100 µg/mL) a precipitation was visible in the culture medium. The two highest concentrations proved toxic. The highest usable concentration was found to be 60 µg/mL. DMSO was used as vehicle. Freshly isolated hepatocytes from a male, adult rat were used. 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 DMN (100 mM) yielded a mean value of 20.1 silver grains per nucleus. This value differs greatly from the two negative controls, by factors of 13.7 and 15.6. The study report contains a Quality Unit statement. It is valid without restriction.


 


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, K2). 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, K2). 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.


 


Ames test:


Polychloro copper phthalocyanine was negative in 2/3 Ames tests with and without metabolic activation (MHLW 2000, K1; BASF 1988, K2). In the other study, the test substance had a weak positive effect in S. typhimurium TA98 with metabolic activation only (Zeiger 1988, K2)


 


In vitro cytogenicity:


Polychloro copper phthalocyanine was negative in CHL/IU cells with and without metabolic activation (Mitsubishi 2000, K1).


 


In an MNT study (BASF 2001, K1) the test substance did not cause any increase in the number of cells containing micronuclei either without S-9 mix or after adding a metabolising system. These findings were confirmed in two modified versions of the in vitro micronucleus assay carried out independently of each other. Thus, under the experimental conditions of this assay, the test substance is considered not to be a chromosome-damaging (clastogenic) agent nor does it induce numerical chromosomal aberrations (aneugenic activity) in-vitro in V79 cells.


 


In vitro gene mutation in mammalian cells:


Polychloro copper phthalocyanine was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro (BASF 2021, K1). The test substance. Four independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and b-naphthoflavone induced rats (exogenous metabolic activation). Since the test substance is a nanoparticle, dose selection for genotoxicity testing was based on the SOP for Preparing Batch Dispersions for in vitro and in vivo Toxicological Studies” of the NANOGENOTOX-Project (Grant Agreement No 2009 21 01); Version 1.2, dated 12 June 2018. Furthermore, to fulfill the requirements of the OECD Guidelines for the HPRT assay, the top concentrations in all main Experiments were defined as the highest homogenous suspension. 0.05% w/v bovine serum albumin water (BSA-water) was used as vehicle. Test groups printed in bold type were evaluated for gene mutations, except the highest tested concentration of the 1st and 2nd Experiment. 2000.0 µg/mL was an inhomogenous suspension and thus is regarded as invalid (data not shown, however archived in the raw data of this study). In the 3rd and 4th Experiment 256.00 µg/mL was used as top concentration which was the highest homogeneous suspension which could be applied to the test culture. 1 st Experiment without S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 1 st Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 2 nd Experiment without S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 2 st Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 2000.00 µg/mL. 3 rd Experiment without S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL. 3 rd Experiment with S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL. 4 th Experiment without S9 mix: 0; 0.125; 0.25; 0.50; 1.00; 10.00; 25.00; 50.00; 100.00; 256.00 µg/mL. Following attachment of the cells for 20 - 24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6 - 8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control substances, ethyl methanesulfonate (EMS) and 7,12-dimethylbenz[a]- anthracene (DMBA), led to the expected statistically significant increase in the frequencies of forward mutations. In all experiments in the absence and the presence of metabolic activation no relevant cytotoxicity (relative survival below 20%) was observed up to the highest concentrations evaluated for gene mutations Based on the results of the present study, the test substance did not cause any biologically relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in four experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.


 


CAS No. 574-93-6


GENOTOXICITY IN-VITRO


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


 


Ames test:


Heliogen Blue MFA was not mutagenic in a preincubation in a standard plate Ames test (tested up to 5000 μg/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 TG 471, BASF AG 2000, K1).


 


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 TG 471, BASF AG 1993, K2). 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 TG 471, BASF AG 1993, K2).


 


Another study with also provided negative results for polychloro copper phthalocyanine in the Ames test (BASF AG 1993, K2).


 


 


CAS No. 27614-71-7:


GENOTOXICITY IN-VITRO


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


 


Ames test:


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 TG 471, BASF 2001, K1).


 


CAS No. 68512-13-0


GENOTOXICITY IN-VITRO


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


 


Ames test:


The test substance was negative in three Ames tests in TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 strains of bacteria with and without metabolic activation (BASF 2005, K1)


 


In vitro cytogenicity:


The effects on chromosomal structure of exposure to the test substance were investigated in cultured CHL (Chinese hamster lung) cells (DIC 1997, K1). 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 of test suspensions (in culture medium) to 24-hour cultures. Cell division was arrested by the addition of the 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 of the test procedure, and the metabolic activity of the S-9 mix employed.

Justification for classification or non-classification

Based on the available data classification for genetic toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.