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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

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Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 25th to June 6th, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21 Jul 1997
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
19 May 2000
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
The Salmonella strains were obtained from KNOLL Aktiengesellschaft on 30 Oct 1989. The Escherichia coli strain was obtained from Merck on 09 Sep 1991.The Salmonella strains are checked for the following characteristics at regular intervals: deep rough character (rfa), UV sensitivity (Δ uvrB) and ampicillin resistance (R factor plasmid).E. coli WP2 uvrA is checked for UV sensitivity.Histidine and tryptophan auxotrophy is automatically checked in each experiment via the spontaneous rate.
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9 mix
Test concentrations with justification for top dose:
1st Experiment - standard plate test: 0, 23, 115, 575, 2875 and 5750 μg/plate
2nd Experiment - preincubation test: 0, 23, 115, 575, 2875 and 5750 μg/plate for TA 1535, TA100, TA 98, E.coli; 0, 12, 60, 300, 1500 and 3000 μg/plate for TA 1537.
3rd Experiment - preincubation test: 0, 2, 10, 50, 250 and 500 μg/plate for TA 1535, TA 100; 0, 0.4, 2, 10, 50 and 100 μg/plate for TA 1537, TA 98.
Vehicle / solvent:
- Vehicle used: DMSO
- Test item preparation: to achieve a solution in the vehicle, the test substance preparation was treated with ultrasonic waves and was shaken thoroughly. All test substance formulations were prepared immediately before administration.
- Justification for choice of vehicle: due to the limited solubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
with and without S9 mix only contains the vehicle
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylendiamine (NOPD)
Details on test system and experimental conditions:
BACTERIA CULTURES
For testing, deep-frozen (-70 °C to -80 °C) bacterial cultures are thawed at room temperature, and 0.1 ml of this bacterial suspension is inoculated in nutrient broth solution (8 g/l Difco nutrient broth + 5 g/l NaCl) and incubated in the shaking water bath at 37 °C for about 12 - 16 hours. As a rule, a germ density of > 10^8 bacteria/ml is reached. These cultures grown overnight are kept in iced water from the beginning of the experiment until the end in order to prevent further growth.

METHOD OF APPLICATION: standard plate incorporation and preincubation

STANDARD PLATE TEST - 1st Experiment
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., 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 mlsolution D (casein solution)
After incubation at 37°C for 48 - 72 hours in the dark, the bacterial colonies (trp+ revertants) are counted.

Replicates: 3 test plates per dose or per control.

PREINCUBATION TEST - 2nd and 3rd Experiment
The experimental procedure is based on the method described by Yahagi et al. and Matsushima et al.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.Replicates: 3 test plates per dose or per control.

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 10^-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: 10^-6), 0.5 ml S9 mix
In the preincubation test, 0.1 ml of the overnight cultures is diluted to 10^-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.
Evaluatrion: the titer is generally 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.

EXOGENOUS METABOLIC ACTIVATION
S9 fraction
The S9 fraction is prepared according to Ames et al. (1, 2). At least 5 male Sprague-Dawley rats (200 - 300 g; Charles River Laboratories Germany GmbH) receive a single intraperitoneal injection of 500 mg per kg body weight Aroclor 1254 (as a 200 mg/ml solution in corn oil) 5 days before sacrifice.
During this time, the animals are housed in Makrolon cages: central air conditioning with a fixed range of temperature of 20 - 24 °C and a relative humidity of 30 - 70 %. The day/night rhythm is 12 hours (light period from 6.00 - 18.00 hours and dark period from 18.00 - 6.00 hours).
Standardized pelleted feed and tap water from bottles are available ad libitum. Five days after administration, the rats are sacrificed, and the livers are prepared (all preparation steps for obtaining the liver microsome enzymes are carried out using sterile solvents and glassware at a temperature of +4 °C). The livers are weighed and washed in a weight-equivalent volume of a 150 mM KCl solution, then cut into small pieces and homogenized in three volumes of KCl solution. After centrifugation of the homogenate at 9000 x g for 10 minutes at +4 °C, 5 ml portions of the supernatant (so-called S9 fraction) are stored at -70 °C to -80 °C.

S9 mix
The S9 mix is prepared freshly prior to each experiment for this purpose, a sufficient amount of S9 fraction is thawed at room temperature and 1 volume of S9 fraction is mixed with 9 volumes of S9 supplement (cofactors). This preparation, the so-called S9 mix, is kept on ice until used. The concentrations of the cofactors in the S9 mix are: MgCl2 8 mM KCl 33 mM Glucose-6-phosphate 5 mM NADP 4 mM Phosphate buffer (pH 7.4) 15 mMThe phosphate buffer is prepared by mixing an Na2HPO4 solution with an NaH2PO4 solution in a ratio of about 4 : 1.To demonstrate the efficacy of the S9 mix in this assay, the S9 batch was characterized with benzo(a)pyrene.

DETERMINATION OF CYTOTOXICITY
oxicity detected by a- decrease in the number of revertants- clearing or diminution of the background lawn (= reduced his- or trp- background growth)- reduction in the titeris recorded for all test groups both with and without S9 mix in all experiments.

SOLUBILITY
Precipitation of the test material is recorded. As long as precipitation does not interfere with the colony scoring, 5 mg/plate is generally selected and analyzed (in cases of nontoxic compounds) as the maximum dose at least in the 1st Experiment even in the case of relatively insoluble test compounds to detect possible mutagenic impurities. Furthermore, doses > 5 mg/plate might also be tested in repeat experiments for further clarification/substantiation.
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 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 were within the historical negative controlrange under all experimental conditions in at least two experiments carried out independently of each other.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
bacteriotoxic effect depending on the strain and test conditions
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
MUTAGENICITY
The test substance did not lead to an increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay).

TOXICITY
A weak bacteriotoxic effect (slight decrease in the number of his+ or trp+ revertants) was occasionally observed in the standard plate test depending on the strain and test conditions from about 575 μg/plate onward.In the preincubation assay strong bacteriotoxicity (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 10 μg/plate onward using the Salmonella strains.
For the strain E. coli WP2uvrA weak bacteriotoxicity (reduced trp- background growth, decrease in the number of trp+ revertants) was observed at 5750 μg/plate in the presence of S9 mix only. Besides a clear reduction in the titer was observed from 2875 μg/plate onward in the presence of S9 mix.

SOLUBILITY
Test substance precipitation was found from about 250 μg/plate onward with and without S9 mix.

CONTROLS
Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria.In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Standard plate test

Dose Without metabolic activation With metabolic activation
Mean (SD) Mean (SD)
DMSO 17 (3) 20 (3)
TA 1535 23 µg 20 (4) 19 (6)
115 µg 19 (2) 17 (2)
575 µg* 16 (4) 17 (8)
2875 µg * 17 (2) 15 (2)
5750 µg * 19 (2) 12 (2)
MNNG 5.0 µg 908 (38)
2-AA 2.5 µg 118 (19)
DMSO 106 (4) 117 (6)
TA 100 23 µg 113 (12) 110 (5)
115 µg 118 (7) 110 (3)
575 µg* 106 (13) 112 (11)
2875 µg * 108 (5) 108 (4)
5750 µg * 101 (2) 95 (5)
MNNG 5.0 µg 1110 (160)
2-AA 2.5 µg 1118 (88)
DMSO 12(4) 11 (1)
TA 1537 23 µg 8 (1) 10 (2)
115 µg 9 (1) 8 (3)
575 µg* 9 (2) 12 (2)
2875 µg * 5 (2) 8 (1)
5750 µg * 4 (3) 7 (2)
AAC 100 µg 376 (29)
2-AA 2.5 µg 154 (15)
DMSO 28 (3) 41 (2)
TA 98 23 µg 28 (2) 37 (5)
115 µg 31 (2) 31 (4)
575 µg* 29 (3) 29 (3)
2875 µg * 37 (6) 30 (3)
5750 µg * 28 (4) 24 (5)
NOPD 10 µg 629 (38)
2-AA 2.5 µg 644 (52)
DMSO 36 (2) 47 (3)
E. Coli WP2 uvrA 23 µg 30 (6) 47 (3)
115 µg 32 (3) 42 (7)
575 µg* 31 (4) 44 (7)
2875 µg * 34 (4) 38 (8)
5750 µg * 35 (4) 42 (3)
4-NQO 5 µg 684 (81)
2-AA 60 µg 264 (13)

* Precipitation

PREINCUBATION TEST - 01

Dose Without metabolic activation With metabolic activation
Mean (SD) Mean (SD)
DMSO 16 (2) 16 (3)
TA 1535 23 µg 12 (2) 14 (3)
115 µg 12 (4) 11 (2)
575 µg* 7 (2) 9 (3)
2875 µg * - -
5750 µg * - -
MNNG 5.0 µg 733 (34)
2-AA 2.5 µg 124 (14)
DMSO 102 (12) 117 (13)
TA 100 23 µg 89 (7) 112 (6)
115 µg 77 (8) 106 (7)
575 µg* 32 (11) 83 (15)
2875 µg * - 62 (22)
5750 µg * - -
MNNG 5.0 µg 716 (25)
2-AA 2.5 µg 778 (69)
DMSO 8 (3) 8 (1)
TA 1537 12 µg 4 (2) 9 (4)
60 µg 5 (1) 7 (4)
300 µg* 3 (2) 3 (1)
1500 µg * 3 (1)  3 (2)
3000 µg * - -
AAC 100 µg 450 (22)
2-AA 2.5 µg 124 (14)
DMSO 0 µg 29 (6) 40 (4)
TA 98 23 µg 28 (4) 29 (2)
115 µg 18 (2) 23 (3)
575 µg* 13 (2) 14 (3)
2875 µg * - 6 (2)
5750 µg * - -
NOPD 10 µg 648 (29)
2-AA 2.5 µg 547 (26)
DMSO 37 (4) 37 (3)
E. Coli WP2 uvrA 23 µg 37 (7) 36 (3)
115 µg 36 (9) 35 (5)
575 µg* 38 (4) 37 (4)
2875 µg * 33 (6) 30 (4)
5750 µg * 29 (2) 15 (4)
4-NQO 5.0 µg 547 (26)
2-AA 60 µg 247 (29)

* Precipitation

PREINCUBATION TEST - 02

Dose Without metabolic activation With metabolic activation
Mean (SD) Mean (SD)
DMSO 14 (2) 15 (3)
TA 1535 2 µg 15 (5) 14 (4)
10 µg 13 (2) 13 (3)
50 µg 13 (4) 14 (2)
250 µg * 11 (2) 11 (1)
500 µg * 9 (4) 9 (3)
MNNG 5.0 µg 556 (31)
2-AA 2.5 µg 146 (19)
DMSO 100 (6) 104 (10)
TA 100 2 µg 97 (10) 100 (5)
10 µg 99 (9) 95 (8)
50 µg 106 (4) 98 (5)
250 µg * 80 (14) 76 (6)
500 µg * 55 (18) 94 (8)
MNNG 5.0 µg 723 (46)
2-AA 2.5 µg 763 (106)
DMSO 8 (2) 9 (1)
TA 1537 0.4 µg 9 (2) 8 (3)
2 µg 9 (3) 9 (2)
10 µg 6 (3) 5 (2)
50 µg  6 (1) 6 (2)
100 µg  3 (2) 5 (3)
AAC 100 µg 429 (20)
2-AA 2.5 µg 115 (8)
DMSO 26 (1) 29 (3)
TA 98 0.4 µg 23 (8) 28 (5)
2 µg 26 (3) 27 (2)
10 µg 20 (4) 22 (2)
50 µg  19 (5) 21 (4)
100 µg  12 (3) 19 (3)
NOPD 10 µg 503 (61)
2-AA 2.5 µg 543 (13)
Conclusions:
Under the experimental conditions, the test item is not a mutagenic substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation
Executive summary:

The substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test was performed according to the OECD guideline 471. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA were assessed in three experiments carried out independently of each other (standard plate test and preincubation assay) with and without metabolic activation (Aroclor-induced rat liver S9 mix).

Precipitation of the test substance was found from about 250 μg/plate onward with and without S9 mix.

A bacteriotoxic effect was observed depending on the strain and test conditions from about 575 μg/plate onward in the standard plate test and from about 10 μg/plate onward in the preincubation test.

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 S9 mix or after the addition of a metabolizing system.

Conclusion

The test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay under the experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 18th to June 06th, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Justification for Read Across is explained in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: OECD Test Guideline 487, In Vitro Mammalian Cell Micronucleus Test. Adopted 26th September, 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: B.49, In Vitro Mammalian Cell Micronucleus Test, Commission Regulation (EU) No. 640/2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
The human peripheral blood lymphocytes used for testing were obtained from healthy non smoking females (up to 35 years of age).
Peripheral blood (heparinized) is taken from donors in certified medical laboratory (MeDiLa) in the morning and transported into the test facility as soon as possible
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
First experiment: 500, 1000 and 2000 μg/ml
Second experiment: 250, 500, 1000 µg/ml
Untreated negative controls:
yes
Positive controls:
yes
Remarks:
aneugenicity control (colchicine) serves as the positive control without S9-mix, and the clastogenicity control (cyclophosphamide) is used to test of the adequacy of the metabolic activation system used
Positive control substance:
cyclophosphamide
other: colchicine
Details on test system and experimental conditions:
TREATMENT AND CULTIVATION
50 µl of an appropriate concentration of test substance solution in medium was added to lymphocyte culture (2.5 ml growth medium RPMI-M + cca 150-210 µl human peripheral blood) in the presence and absence of a metabolic activation system (S9-mix).
In the first experiment after shaking the cultures were cultivated with the test substance for 3 hours. After that they were rinsed up by RPMI-M and then transported to the fresh growth medium (RPMI-M) with 11.25 µl of cytochalasin B (the final concentration in cultures was 4.5 µg/ml). Cultures were then cultivated and sampled after 23 hours since the beginning of treatment. The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure.
In the second experiment (prolonged exposition without activation) cultures were treated with test substance without metabolic activation for 23 hours in the presence of cytochalasin B (7.5 µl; final concentration in cultures was 3 µg/ml). No transport to fresh medium was needed in that experimental design.

REPLICATES: duplicate cultures were used for each concentration and control.

CELL CYCLE LENGTH DETERMINATION
The proliferation of lymphocytes was evaluated by doubling time experiment. Four separate tubes with blood in the medium were prepared in day 2. Every following day (day 3, day 4 and day 5) lymphocytes were isolated and quantified. Then the proliferation curve was constructed and cell cycle length was determined. The lymphocyte`s cell cycle length during the first experiment was 12 hours.

SCHEDULE OF THE TEST
Day 1: blood sampling. Blood was taken and then stored in the fridge until the next day.
Day 2 and 3: cultivation. The whole human peripheral blood was transferred to the growth medium and mitogenic stimulator (phytohaemagglutinin M) was added. These operations were carried out in a laminar box at room temperature. The cultivation runs without interrupting for 48 hours (37 ± 1 °C; 5 % CO2).
Day 4: exposition. The test substance, positive and negative control substances were added to the individual cultures (in a laminar box at room temperature). In the first experiment without and with metabolic activation (S9-mix), the cultures were washed (after 3 hours of continuous exposure to the test substance in 37 ± 1 °C; 5 % CO2) and transferred to fresh culture medium with cytokinesis blocker (cytochalasin B). Washing and transfer were carried out in a laminar box at room temperature. Then the lymphocytes were cultured (37 ± 1 °C; 5 % CO2) for remaining period (total 23 hours from the start of exposure).
Day 5: harvesting of cultures. All cultures were processed in a laminar box at room temperature, (with hypotonia, fixation solution). Suspensions were dropped on clear microspcopic slides. The slides were allowed to dry at least overnight.
Day 6: staining of slides. Slides were stained by Giemsa-Romanowski staining solution.

PREPARATION OF SLIDES
Cultures were harvested 23 hours after the beginning of treatment (after about 1.5 to 2 cell cycles). Cultures were treated by hypotonic solution (RT, ca 5 min.) and then they were centrifuged (1200 rpm, 10 min.). After removing of hypotonic solution, fixation solution was added to cultures and cultures were centrifuged again (1200 rpm, 10 min.). The addition of fixation solution and centrifugation were repeated three times. Suspensions were then dropped on clear microscopic slides. Preparations were let to dry at laboratory temperature and then slides were stained by Giemsa Romanowski staining solution.

SCORING OF CELLS FOR CYTOTOXICITY AND GENOTOXICITY EVALUATION
All slides were coded before microscopic analysis.
At least 1000 cells were scored per each concentration and controls divided equally between the duplicates for determination of cytotoxicity. CBPI index was calculated from ratio of mononucleated, binucleated and multinucleated cell at each culture. The cytotoxic effect was characterized as % of cytotoxicity.
2000 binucleated cells were analysed per each concentration and control divided equally between the duplicates for determination of genotoxicity. The genotoxic effect is characterized by numbers of binucleated cells with micronuclei.

ACCEPTABILITY OF EXPERIMENT RESULTS
Results are accepted if:
- numbers of binucleated cells with micronuclei in negative and positive controls are in actual ranges of historical controls
- at least 1000 binucleated cells per culture are available for scoring at each concentration (if there is clearly positive results, the number of scoring cells could be lower).
- at least 3 concentrations are analysable.

DOSE SELECTION
At first the cytotoxicity of test substance has been determined by measuring of cell proliferation. In the highest concentration (2000 µg/ml) no cytotoxicity or precipitate was observed; test item did not cause a marked change in the pH of the medium (in used concentrations).
Cytotoxicity test concentrations:
First experiment 125, 250, 500, 1000 and 2000 µg/ml
Second experiment 250, 500, 1000 and 2000 µg/ml
The highest concentration 2000 µg/ml could not be used for genotoxicity evaluation because of high cytotoxicity in this exposure time (23 hours).

METABOLIC ACTIVATION SYSTEM
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors. The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/mL, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames (1983). The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique at a temperature below –70 °C. Fresh solution of the cofactors (1.6 mM MgCl2.6H2O, 0.8 mM NADP and 1 mM glucose-6-phosphate) was prepared before each experiment with metabolic activation. Each culture in experiments with metabolic activation contained 18.5 µl of S9 and 18.5 µl of cofactors solution (S9-mix).
Evaluation criteria:
Genotoxic potential is indicated by increasing of number of binucleated cells with micronuclei in comparison to the negative control (two-fold increase rule) and by dependence of number of binucleated cells with micronuclei on dose (dose-response relationship).
Ratio of number of binucleated cells with micronuclei at tested concentration to number of binucleated cells with micronuclei in negative control (Mt/Mc) should be higher than 2. The result will be considered as positive when two-fold increase rule will be met at least one tested concentrations.
Test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control (two-fold increase rule)
- the dependence of increasing number of cells with micronuclei on concentration (dose-response relationship) is evident
- any of the results are outside the distribution of the historical negative control data

Test chemical is considered clearly negative if, in all experimental conditions examined:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data
In case of equivocal results, further testing with modification of experiment conditions will be used for clarification.
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The first experiments gave negative results, so the second experiment without metabolic activation had to be done with extended exposure (23 hours) in the presence of cytochalasin B. The results did not show substantial (biologically significant) increase in the number of binucleated cells with micronuclei (Mt/Mc > 2, two-fold increase).

Actual results of negative and positive controls fulfilled the acceptability criteria - the actual values did not exceed limits of historical controls in the testing laboratory.

CYTOXICITY
All of test concentrations did not show the cytotoxicity higher than 55 ± 5 % in the time of exposure 3 hours. Test concentration 2000 µg/ml did show the cytotoxicity in the prolonged exposition without activation (23 hours). Other used concentrations 1000, 500 and 250 µg/ml did not show the cytotoxicity in the prolonged exposition.
On the basis of these results, the concentration of 2000 µg/ml was selected as the highest concentration for the analysis of genotoxic effect in the time of exposure 3 hours. For the analysis of genotoxic effect in the prolonged time of exposure 23 hours was selected as the highest concentration 1000 µg/ml (second experiment).
Conclusions:
Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.
Executive summary:

In Vitro Mammalian Cell Micronucleus Test was performed according to OECD Test Guideline No. 487.

The human peripheral blood lymphocytes from healthy donors were used for testing. The test substance was suspended in RPMI medium and assayed in five concentrations 125 - 2000 µg/ml, which were applied to cultures in volume of 50 µl.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Conclusion

Under the experimental design described, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From January 13th to April 07th, 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Justification for Read Across is explained in the endpoint summary and it is further detailed in the report attached to the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted July 28th, 2015
Deviations:
yes
Remarks:
no impacting the outcome of the study
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
yes
Remarks:
no impacting the outcome of the study
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Source: frozen permanent cell culture was obtained from European Collection of Cell Cultures (ECACC).
- Breeding: the cells were kept at -196 ºC under liquid nitrogen. After activation, cells are grown in DMEM medium with L-glutamine and 10 % FBS in incubator (5 % CO2, 37±1 °C, moistened). Cells underwent maximum 7 passages after thawing the original culture delivered from cell collection before using for test.
- Cleaning: cleansing of cultures was performed 5 days before treatment with complete medium supplemented with HAT supplement due to elimination of mutants.
- Mycoplasma determination: cell cultures were checked for mycoplasma contamination. At every experiment one withdrawal of media has been performed and sent to the contract laboratory performing mycoplasma determination.
- Cell cycle duration: duration of cell cycle was determined according to ATCC Animal Cell Culture Guide, Tips and techniques for continous cell lines.)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of rat liver homogenate and mixture of cofactors
Test concentrations with justification for top dose:
EXPERIMENT: 0.25, 0.5, 1.0, and 2.0 mg/ml
CYTOTOXICITY: 0.0625, 0.125, 0.25, 0.5, 1 and 2 mg/ml
Vehicle / solvent:
The test substance is soluble in water so it was tested dissolved in Dulbecco's minimal essencial medium (DMEM).
Untreated negative controls:
yes
Remarks:
negative control plates contained 10 ml of complete medium
Negative solvent / vehicle controls:
yes
Remarks:
plates (DMSO) contained 9.9 ml of completemedium and 0.1 ml of DMSO
Positive controls:
yes
Remarks:
plates contained 9.9 or 9.95 ml of complete medium and 100 or 50 µl of relevant positive control diluted in DMSO
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
APPLICATION
Every dish with negative control/test substance contained 1.0 ml of application form of the test substance in DMEM so that the final concentrations on dishes were as given above and 9.0 ml of complete medium or activation mixture (S9 mix).

DURATION and SCHEDULE
- Treatment: cells were treated for 3 hours (with as well as without metabolic activation; day 1).
- Seeding: after treatment, approximately 2x10^6 cells were transferred to suitable number of dishes to seed enough cells.
- Determination of plating efficiency: cells were seeded for detection of number of cells (PE estimation).
- Subculturing: on the 3rd, 6th and 8th day, approximately 2x10^6 cells from every culture were transferred and 10th day. The cell populations were subcultured in order to maintain them in exponential growth.
- Extraction of mutants: extractions of mutants was performed with using selective medium together with PE estimation again.

DETERMINATION OF SURVIVAL
After treatment period, the cultures were trypsinised and an aliquot (0.3 ml of 10^3/ml cell suspension) was diluted and plated to 6 cm Petri dishes to estimate the viability of the cells. A number of cells were then replaced in order to maintain the treated cell populations; the number of cells taken forward was adjusted according to the expected viability of the cultures, to give two millions of viable cells. Cells were grown in 10 cm Petri dishes.

INCUBATION
Survival and plating efficiency plates were incubated for at least six days (37±1 ºC, 5 % CO2, moistened) prior to scoring. Mutant plates were incubated for an appropriate period to ensure adequate colony size (about 10 days). After incubation, the plates were stained with methylene blue and colonies were scored.

NUMBER OF REPLICATIONS
Every concentration was tested in two independent runs.

DETERMINATION OF MUTANT FREQUENCY
At expression time, each culture was trypsinised, resuspended in complete medium and counted by microscopy. Then the following procedures were performed:
- an adequate number of cells were subcultured to maintain the treated populations of cells. This step is not performed on the 10th day.
- after dilution, an estimated 220000 cells were plated in each of ten 100 mm tissue culture Petri dishes (together 2200000 cells). After 1 hour, 6-thioguanine was added to each the Petri dish to final concentration of 5 µg/mL. Only HPRT mutant colonies are able to grow in the presence of 6-thioguanine; these plates were subsequently scored for the presence of mutants.
- after dilution, an estimated 300 cells were plated in each of three 60 mm tissue culture Petri dishes. These plates were used to estimate plating efficiency.

ASSSAY ACCEPTANCE CRITERIA
- Concurrent negative controls should be within the 95 % control limits of the distribution of the laboratory’s historical negative control database.
- Concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
- Two experimental conditions (i.e., with and without metabolic activation) were tested unless one resulted in positive results.
- Adequate number of cells is used and concentrations are analysable.


CYTOTOXICITY
The test substance was dissolved in DMEM at the maximum concentration of 20.0 mg/ml. This concentration was dissolved according to recommendation given in OECD TG 476 to three other lower concentrations. These concentrations were 2.5, 5.0, and 10.0 mg/ml.

METABOLIC ACTIVATION SYSTEM
The metabolic activation was performed by S9 fraction of rat liver homogenate and mixture of cofactors.
The liver homogenate was prepared from Wistar male rats weighing approximately 200 g, previously induced with Delor 106 (a mixture of PCBs). Delor 106 was diluted with olive oil to a concentration of 200 mg/ml, and each rat was administered a single injection of 500 mg/kg 5 days before S9 preparation. The S9 was prepared according to the methods described by Maron and Ames. The liver was removed from each animal and washed in ice cold 0.15 M KCl. The livers washed were mixed with another 0.15 M KCl (3 ml/g wet liver) homogenized in a grinder, and the tissue suspension was centrifuged for 10 min at 9000 g. Aliquots of the supernatant (S9) were stored in plastic tubes using sterile technique
at a temperature below –70 °C.

Every lot of S9 was tested for sterility and activity in the Ames test with the aid of bacterial strain TA 98. Activity was within expected limits.
Cofactors (NADP and glucoso-6-phosphate) were dissolved in PBS. Composition of S9 mix was as follows:
S9 tissue fraction 1.0 ml
NADP (0.1M) 0.4 ml
G-6-P (0.1M) 0.5 ml
KC1 (0.33M) 1.0 ml
MgCl2 (0.1M) 0.5 ml
Phosphate Buffer (0.2 M) 4.6 ml
Each plate in all experiments with metabolic activation contained 4 ml of S9mix, 5 ml of complete medium and 1.0 ml of the test substance solution.
Evaluation criteria:
Each experiment is separately evaluated using modified two-fold increase rule according to Claxton L.D. et al, Mutat. Res.,189, 83-91, 1987.
The mutagenic potential is indicated by increasing number of mutants in treated groups in comparison to the negative solvent control (modified two-fold increase rule and any of the results outside the distribution of the historical negative control data) and/or by dependence of increasing number of mutants on dose (dose-response relationship).
There is no requirement for verification of a clearly positive or negative response.
In cases when the response is neither clearly negative nor clearly positive than a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions i.e. S9 concentration or S9 origin) could be performed.
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:
MUTAGENICITY
Mutation frequency of cells treated with the test substance in all experiments was low in all concentrations; Mt/Msc ratio generally did not exceeded 3 fold limits. At the same time, no dose dependence was observed in any experiment.
As nor the dose-dependence neither repeated 3-fold increase was reached we do not consider these single increased values biologically significant.

Mutation frequency of negative controls varied from 0.77 – 1.53 mutants per 10^5 plated cells. Historical control range is 0.0-3.42 mutants per 10^5cells.
Mutation frequency of positive controls was sufficiently high:
EMS 50 µl 11.14, historical control range is 6.49 – 21.75 mutants per 10^5 cells
EMS 100 µl 15.10, historical control range is 12.43 – 46.34 mutants per 10^5 cells.
DMBA 19.91 and 22.68, historical control range is 0 – 46.34 mutants per 10^5 cells

PLATING EFFICIENCY
For the assessment of number of plated cells, PE was determined always after treatment. In all concentrations used in both experiments more than two millions cells were influenced in single replicates.
In most cases more than two millions of cells were grown for expression of mutants. It does not apply to treatment with metabolic activation 2.0 mg/l, replicate 2, where less than two million cells were transferred to further growth after treatment. Another such case was the dose of 1.0 mg/ml in experiment with metabolic activation (replicate 1) where 3 dishes in the last passage were contaminated with mould. In this case only cells from the other 4 dishes were processed. Both the doses are marked with cursive letters.

CYTOTOXICITY
Treatment with the test substance for 3 hours resulted in no toxicity.
On the basis of the result obtained, the concentration of 0.5 mg/ml was selected as the highest concentration to be used in the mutation assays-. The lower concentrations were spaced by a factor approximately 2-√10, in order to reach a concentration row.

MYCOPLASMA DETERMINATION
Results of both test samples were negative so all media after cultivation of cells were free of mycoplasma.

MUTAGENICITY

Summary of results: 3-hour treatment without metabolic activation

Conc. (mg.ml) MF/105 Mt/Msc
NC 0.88 1.00
0.25 0.74 0.84
0.5 0.5 0.57
1 0.68 0.77
2 0.75 0.85
EMS50 11.14 12.6
EMS100 15.1 17.08

Summary of results: 3-hour treatment with metabolic activation

Conc. (mg.ml) MF/105 Mt/Msc
NC 1.33 1.00
0.25 1.38 1.04
0.5 1.24 0.93
1 1.17 0.88
5 0.73 0.55
DMBA 21.14 15.93

Mutagenicity without metabolic activation, 3-hour treatment - extraction time 9 days

Conc. (mg/ml) Viability (number of colonies) avg PE (%) Mutants (number of colonies) ∑M NPC MF/105 cells Mt/Msc
NC1 439 437 398 425 102 2 2 6 2 4 3 2 4 2 4 31 3114222 1 1.13
NC2 440 366 419 408 98 4 4 1 2 1 1 3 2 3 2 23 2994444 0.77 0.87
0.25 (1) 445 432 459 445 106.9 0 3 3 3 4 6 2 2 2 0 25 3265778 0.77 0.87
0.25 (2) 485 493 492 490 117.6 5 3 2 2 0 3 2 3 2 4 26 3593333 0.72 0.82
0.5 (1) 358 415 392 388 93.2 2 2 2 0 1 2 4 0 0 0 13 2847778 0.46 0.52
0.5 (2) 424 413 373 403 96.8 1 1 3 2 1 1 2 1 3 1 16 2957778 0.54 0.61
1.0(1) 438 445 483 455 109.3 1 1 4 0 2 2 1 1 2 4 18 3339111 0.54 0.61
1.0(2) 345 368 396 370 88.8 4 1 3 3 2 4 1 2 2 1 23 2710889 0.85 0.96
2.0(1) 375 375 326 359 86.1 3 3 1 2 1 1 3 1 0 3 18 2630222 0.68 0.77
2.0(2) 336 307 306 316 76.0 2 1 3 1 3 1 1 3 3 1 19 2319778 0.82 0.93
EMS50 343 325 346 338 81.2 18 27 33 31 22 26 36 32 22 29 276 2478667 11.14 12.6
EMS100 507 525 507 513 123.2 51 47 53 67 57 61 62 63 59 48 568 3762000 15.1 17.08

Mutagenicity with metabolic activation, 3-hour treatment - extraction time 9 days

Conc. (mg/ml) Viability (number of colonies) avg PE (%) Mutants (number of colonies) ∑M NPC MF/105cells Mt/Msc
NC1 335 336 362 336 92.2 3 3 3 1 4 2 1 5 1 4 27 2460333 1.1 0.83
NC2 363 365 423 384 105.4 4 5 1 5 3 3 5 6 7 4 43 2813556 1.53 1.15
0.25 (1) 363 372 375 370 101.6 1 4 4 3 1 5 4 3 4 0 29 2713333 1.07 0.81
0.25 (2) 276 294 300 290 79.7 4 3 3 5 3 2 1 6 6 5 38 2126667 1.79 1.35
0.5 (1) 339 308 325 324 89 3 4 2 1 1 1 6 2 4 1 25 2376000 1.05 0.79
0.5 (2) 271 288 289 283 77.7 4 6 3 3 1 2 1 2 3 5 30 2072889 1.45 1.09
1.0(1) 374 367 365 369 101.3 3 4 2 3 4 3 2 4 1 2 28 2703556 1.04 0.78
1.0(2) 370 421 384 392 107.6 7 3 0 3 3 4 2 4 5 6 37 2872222 1.29 0.97
2.0(1) 386 407 384 392 107.8 2 3 4 2 4 3 6 1 2 2 29 2877111 1.01 0.76
2.0(2) 384 435 429 416 114.3 1 2 0 1 0 4 2 1 2 1 14 3050667 0.46 0.35
DMBA 373 408 378 386 106.1 60 52 42 39 51 69 70 74 50 57 564 2833111 19.91 15
DMBA 326 311 290 309 84.9 51 55 57 65 49 60 59 41 38 39 514 2266000 22.68 17.09

PLATING EFFICIENCY

Plating efficiency after 3-hour treatment and number of survived cells – experiment without metabolic activation

Conc. (mg/ml) Viability (number of colonies) mean PE (%) ∑ NSC NPC ∑ NPC
NC1 307 269 283 286 96.9 8.47E+06 2.34E+06 4.83E+06
NC2 274 303 337 305 103.1 7.27E+06 2.49E+06
0.25 (1) 278 242 279 266 90.1 1.01E+07 2.18E+06 4.27E+06
0.25 (2) 270 239 260 256 86.7 1.11E+07 2.09E+06
0.5 (1) 287 250 254 264 89.2 8.53E+06 2.15E+06 5.18E+06
0.5 (2) 396 360 356 371 125.4 7.13E+06 3.03E+06
1.0(1) 300 312 282 298 100.8 1.01E+07 2.43E+06 4.99E+06
1.0(2) 295 296 347 313 105.8 8.00E+06 2.55E+06
2.0(1) 374 402 363 380 128.5 1.22E+07 3.10E+06 6.27E+06
2.0(2) 357 411 396 388 131.3 8.80E+06 3.17E+06
EMS50 858 850 910 873 295.3 1.17E+07 7.13E+06 -
EMS100 304 326 367 332 112.5 9.27E+06 2.71E+06 -

Plating efficiency after 3-hour treatment and number of survived cells – experiment with metabolic activation

Conc. (mg/ml) Viability (number of colonies) mean PE (%) ∑ NSC NPC ∑ NPC
NC1 261 249 255 255 93.5 4800000 2080000 4450000
NC2 297 284 290 290 106.5 3870000 2370000
0.25 (1) 258 220 278 252 92.4 7930000 2060000 4550000
0.25 (2) 279 321 315 305 111.9 7000000 2490000
0.5 (1) 250 222 250 241 88.3 7400000 1970000 4020000
0.5 (2) 250 253 250 251 92.1 6900000 2050000
1.0(1) 261 270 276 269 98.7 7150000 2200000 4420000
1.0(2) 246 291 281 273 100 7300000 2230000
2.0(1) 248 244 274 255 93.6 8600000 2090000 3760000
2.0(2) 218 217 181 205 75.3 7600000 1680000
DMBA 255 264 281 267 97.8 5800000 2180000 4180000

CYTOTOXICITY

Toxicity test without metabolic activation, 3-hour treatment

Concentration (mg/ml) Viability (number of cells per plate) avg PE
DMEM 332 337 347 339 100
0.0625 466 475 500 480 141.8
0.125 422 456 415 431 127.3
0.25 331 304 321 319 94.1
0.5 366 351 330 338 99.8
1 332 356 297 328 96.9
2 365 342 397 368 108.7
Conclusions:
Under the experimental conditions, the test substance was non-mutagenic for V79 cells with as well as without metabolic activation.
Executive summary:

The test substance was assayed for the mutagenicity by the In Vitro Mammalian Cell Gene Mutation Test. The performed test was based on OECD Test Guideline No. 476 – In Vitro Mammalian Cell Gene Mutation Test (2015), which is analogous to the EU method B.17. V79 hamster fibroblast were used for testing. The test substance was dissolved in DMEM in concentration 2.0 mg/ml. Concentrations tested for cytotoxicity in 3-hour experiment were 2.0, 1.0., 0.5, 0.25, 0.125 and 0.0625 mg/ml. No toxicity was observed in any dose.

Mutagenicity experiment (3 hour treatment) with the test substance followed then. Four concentrations of test substance - 2.0, 1.0, 0.5 and 0.25 mg/ml - were used. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation.

In the arrangement given above, the test substance was non-mutagenic for V79 cells without as well as with metabolic activation.

Conclusion

The test substance was non-mutagenic for V79 cells without as well as with metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Evaluation of the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro Ames tests, in vitro gene mutation on mammalian cells and in vitro chromosomal aberration.

An AMES test is available on Basic Blue 140 (BBl140), however there are no data regarding mammalian cell gene mutation, neither chromosomal aberration, thus the available data on structural analogues Similar Substance 02 and Similar Substance 01 have been taken into account.

BBl140, Similar Substance 02 and Similar Substance 01 share the same phthalocyaninic scaffold structure. BBl140 and Similar Substance 01 present a zwitterionic moiety formed by sulphonic acid, which conducts an acid function, and a tertiary amine on the sulphonaminic chain, characterized by a basic function. BBl140 presents a further sulphonaminic functionalization, of which chain ends with a tertiary amine that, based on the stechiometrical ratio reported in the analytical characterization, in most of the cases can be salified with acetic acid.

Similar Substance 02 is functionalized by sulphonamine and two sulphonic acids, only in one case salificated by sodium.

Structural differences are expected to not significantly impact the genotoxicity potential. The read across approach has been further detailed in the report attached to the IUCLID section 13.

BACTERIA GENE MUTATION ASSAY

BBl140 was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. The test was performed according to the OECD guideline 471. Salmonella typhimurium strains TA 1535, TA 100, TA 1537, TA 98 and Escherichia coli WP2 uvrA were assessed in three experiments carried out independently of each other (standard plate test and preincubation assay) with and without metabolic activation (Aroclor-induced rat liver S9 mix), involving the combination of strains recommended by OECD guideline. Precipitation of the test substance was found from about 250 μg/plate onward with and without S9 mix. A bacteriotoxic effect was observed depending on the strain and test conditions from about 575 μg/plate onward in the standard plate test and from about 10 μg/plate onward in the preincubation test. 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 S9 mix or after the addition of a metabolizing system, thus the test substance has been judged not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay.

A comparable behaviour has been observed in the AMES test conducted using the Similar Substance 01. Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 were assayed with the test item by a method similar to OECD guideline 471. Due to precipitation of the substance during a first experiment, doses ranging from 100 to 3200 µg/tube were chosen for the repeat tests. None of the five strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. Compound precipitation started at 500 µg/plate and 5000 µg/plate could not be used for evaluation. No bacteriotoxic effects were recorded up to 158 µg/plate. Total bacterial count remained unchanged and no growth inhibition was seen. At doses above 158 µg/plate, there was strain-specific cytotoxicity. Under the experimental conditions, the test item resulted to be not a mutagenic.

MAMMALIAN CELL GENE MUTATION ASSAY

Similar Substance 02 was assayed for the mutagenicity following the testing procedures outlined in the OECD Guideline No. 476. V79 hamster fibroblast were used for testing and the concentrations tested for cytotoxicity in 3-hour experiment were 2.0, 1.0., 0.5, 0.25, 0.125 and 0.0625 mg/ml. No toxicity was observed in any dose. Mutagenicity experiment (3 hour treatment) with the test substance followed then; four concentrations of test substance - 2.0, 1.0, 0.5 and 0.25 mg/ml - were used. Each concentration was tested in two independent runs. Experiments were performed with as well as without metabolic activation. The test substance was non-mutagenic for V79 cells without as well as with metabolic activation.

Supporting information are also available on Similar Substance 01. The mutagenic potential of the test substance was investigated according to OECD guideline 476. The test strain were Chinese Hamster Lung fibroblasts (V79). The HPRT gene locus was the target of the test. Two independent experiments were performed in the assay, with and without liver microsomal activation. The highest concentration of the pre-experiment (2000 μg/ml) was limited by the solubility properties of the test item in DMSO and aqueous medium. The maximum concentration of the main experiments was limited by cytotoxic effects and precipitation of the test item. In both main experiments, no substantial and reproducible dose-dependent increase in mutation frequency was observed.

CHROMOSOMAL ABERRATION ASSAY

The chromosomal aberration potential has been investigated taken into account the available information on the structural analogues.

In Vitro Mammalian Cell Micronucleus Test was performed according to OECD Test Guideline No. 487 on Similar Substance 02. The human peripheral blood lymphocytes from healthy donors were used for testing. Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors. Under the experimental conditions, the test substance had no genotoxic effects in the human peripheral blood lymphocytes in experiments both without and with metabolic activation. The result of micronucleus test was negative, test substance is then considered not able to induce chromosome breaks and/or chromosome gain or loss in this test system.

Similar substance 01 was also examined according to OECD guideline 487. Chinese hamster lung fibroblasts (V79) cells was the test strain. Metabolic activation was achieved by S9 mix from Aroclor 1254-induced male Sprague-Dawley rats. At concentrations equal/higher than 10 µg/ml, cytotoxic effects were observed after 4 h and 24 h of treatment. Precipitation in the medium was observed from 50 µg/ml; therefore, concentrations of 10, 25 and 50 µg/ml (without and with S9 mix, 4 h and 24 h) were chosen for reading.

No biologically relevant increase in the frequency of micronucleus-containing cells were observed in the test, at 4 h and 24 h, in the presence or absence of metabolic activation.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggest that test substance did not show any reasons of concern from the genotoxicity point of view.

 

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).