Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (similar to OECD TG 471; non-GLP): negative

UDS test (similar to OECD TG 482; GLP): negative

Chromosmomal aberration test (acc. to Japanese Guidelines for Screening Mutagenicity Testing of Chemicals; GLP compliance not specified): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
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, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
in the standard plate test
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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 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 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 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 conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Mouse spot test (similar to OECD TG 484; GLP): negative

Erythrocyte micronucleus test (similar to OECD TG 474; GLP): negative

Link to relevant study records

Referenceopen allclose all

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

Additional information

GENOTOXICITY IN VITRO:

Gene mutation in bacteria:

The test substance was not mutagenic in a standard plate Ames test (equivalent or similar to OECD TG 471) with and without metabolic activation (tested up to 10000 μg/plate in Salmonella typhimurium TA1535, TA1537, TA98 and TA100; metabolic activation: S9 fraction from the liver of male Sprague-Dawley rats, treated with a single dose of 500 mg/kg bw Aroclor 1254 five days before sacrifice and mixed with a series of cofactors). Cytotoxicity was not observed with or without metabolic activation.

In another Ames test a pre-incubation assay, 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), the test substance was also not mutagenic (JETOC 1995). No cytotoxicity was observed.

DNA damage and/or repair:

UDS assays (equivalent or similar to OECD TG 482; GLP) with rat hepatocytes as well as human fibroblasts were found to be negative for DNA damage/or repair when tested up to precipitating concentrations.

Cytogenicity in mammalian cells:

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

It should be noted that there were several weakly positive Ames test results, when crude copper phthalocyanine was investigated for mutagenic effects in bacteria (Ciba-Geigy 1986 (1x), 1987 (1x) and 1988 (2x), all Val. 3). However, today it is known that these crude copper phthalocyanines were manufactured by using nitrobenzene and chlorobenzene as solvents in the past. Therefore, the test material contained residues of these solvents and these impurities were considered to be responsible for the weakly mutagenic effects observed in vitro. Meanwhile this manufacturing process was replaced by other manufacturing processes which all use alternative solvents.

GENOTOXICITY IN VIVO:

Gene mutation in vivo:

In a mouse spot test similar to OECD TG 484 it was assessed whether the test 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 postnatally by examination of the fur of young mice for recessive spots (RS) resulting from expression of recessive genes involved in coat-colour determination.

The test substance 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 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 bw) performed simultaneously yielded an statistically significant average RS frequency of 4.75 %.

Cytogenicity in vivo:

In a micronucleus test conducted similar to OECD TG 474, the test substance 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. 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 both of these in vivo tests, also the crude test material 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 was obtained.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.