Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was not found to be mutagenic in vitro. The test item was concluded to be negative for chromosome aberrations under the condition tested. Neither AHTN itself nor any metabolite generated by the metabolic activation system is mutagenic in the Ames test under the described experimental conditions. The test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 October - 22 November 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study following OECD guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
The standard plate incorporation and the preincubation methods
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 102
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S-9 mix
Test concentrations with justification for top dose:
Dose range 8-5000 µg/plate.
Vehicle / solvent:
Dimethyl Sulfoxide (Merck)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1535 and TA100
Positive control substance:
sodium azide
Remarks:
SERVA, Lot 30175 - Dosage: 1.0µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA1537 and TA97
Positive control substance:
other: ICR 191 without metabolic activation S9
Remarks:
SERVA, Control K8 - Dosage: 1.0µg/plate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA98
Positive control substance:
2-nitrofluorene
Remarks:
ALDRICH, Lot 3734435 - Dosage: 0.5µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for TA102
Positive control substance:
mitomycin C
Remarks:
SIGMA, Lot 32 H0326 - Dosage: 0.4µg/plate

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for E. coli WP2 uvr A
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
4-Nitroquinolineoxide (SIGMA, Lot 84F-0572)

Migrated to IUCLID6: without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
for all strains to examine the activity of the S9 mix
Positive control substance:
other: 2-Aminoanthracene - with and without metabolic activation S9
Remarks:
SIGMA, Lot 121 H3475
Details on test system and experimental conditions:
The Salmonella typhimurium strains TA1535, TA1537, TA97, TA98, TA100 and TA102 were obtained from B.N. Ames. E. coli WP2 uvrA was obtained from The National Collection of Industrial and Marine Bacteria Ltd, Aberdeen Scotland.
Evaluation criteria:
There was no generally accepted statistical treatment of Ames test and E.coli reversion system data. In most tests the results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of his+ revertants. The increase should reach at least a doubling of the number of spontaneous revertants for Salmonella typhimurium strains TA1535, TA1537, TA98 and E. coli VP2 uvrA. For strains TA97, TA100 and TA1O2 a 1.5 - fold increase over control values might be indicative of a mutagenic effect provided the negative control values fall within the historical control data. Other investigators have set higher limits for a mutagenic response (factor 3 and 2 for the respective groups of strains). These rules of thumb have a questionable scientific foundation and biological relevance should always be taken into account. A negative result is defined as the absence of a reproducible increase in the number of bis+ or trp+ revertant colonies.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The results of the standard plate incorporation test are summarized in table 1 and of the preincubation test in table 2.

The spontaneous mutation rate was within the range observed for historical controls. All positive controls gave acceptable responses.

Toxic effects were not apparent in the standard assay, thus a dose range of 8 to 5000 µg/plate was evaluated to include solutions and suspensions.

AHTN did not cause en increase of the number of revertant colonies in any of the seven tester strains.

Table I Salmonella mutagenicity test (standard assay). Mean values and standard deviations.

Experiment No 01 01 01 01 01 01 02 02 02 02 02 02 02 02
Activation -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Strain TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA TA97 TA97 TA9B TA98 TA100 TA100 TA102 TA102
Concentration
µg/plate
                           
0 13 9 10 10 24 22 245 245 26 28 196 197 312 298
  ±5 ±3 ±4 ±4 ±8 ±6 ±20 ±3 ±6 ± 4 ±21 ±25 ±24 ±7
8 13 7 7 11 23 20 258 257 24 32 197 207 324 292
  ±2 ±3 ±1 ±4 ±5 ±1 ±20 ±11 ±4 ± 7 ±12 ±16 ±20 ±19
40 15 12 9 11 19 26 250 251 27 27 179 208 330 314
  ±1 ±4 ±2 ±5 ±6 ±2 ±16 ±20 ± 4 ± 5 ±15 ± 7 ±18 ± 9
200 13 10 9 9 19 21 252 273 27 36 194 203 312 346
  ±5 ±3 ±1 ±2 ±5 ±5 ±17 ±25 ±4 ±11 ±14 ±17 ±27 ±17
1000 16 8 8 9 15 21 240 231 25 28 197 176 307 291
  ±3 ±2 ±1 ±2 ±2 ±6 ±19 ±30 ± 5 ± 5 ±14 ±24 ±16 ±19
5000 14 6 6 4 21 23 237 234 21 26 203 199 278 319
  ±4 ±4 ±1 ±2 ±4 ±5 ±23 ±20 ± 4 ± 2 ± 8 ± 8 ±13 ±30

Table 2 Salmonella mutagenicity test (Liquid preincubation assay). Mean values and standard deviations.

Experiment No 01 01 01 01 01 01 02 02 02 02 02 02 02 02
Activation -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9 -S9 +S9
Strain TA1535 TA1535 TA1537 TA1537 WP2uvrA WP2uvrA TA97 TA97 TA9B TA98 TA100 TA100 TA102 TA102
Concentration
µg/plate
                           
0 7 8 13 7 27 28 258 265 25 33 162 132 359 398
  ±4 ±2 ±2 ±1 ±8 ±6 ±14 ±10 ± 8 ±10 ±11 ±14 ± 26 ± 31
8 13 7 13 8 28 29 -287 281 24 31 110 125 358 434
  ±2 ±1 ±4 ±2 ±5 ±3 ±15 ±5 ± 6 ± 6 ±13 ± 20 ± 18 ± 22
40 11 4 11 6 35 31 285 276 21 32 98 144 342 423
  ±1 ±1 ±3 ±2 ±9 ±3 ±15 ± 3 ± 4 ±9 ±12 ±21 ± 11 ± 14
200 13 8 10 8 30 32 273 295 27 35 102 107 325 343
  ±3 ±1 ±1 ±5 ±4 ±3 ±28 ±13 ± 2 ± 3 ± 8 ±8 ± 20 ± 17
1000 11 8 9 8 26 35 298 301 23 30 100 105 217 233
  ±2 ±6 ±4 ±3 ± 6 ± 3 ±21 ±27 ± 4 ± 4 ± 7 ± 15 ± 17 ± 26
5000 12 7 11 10 33 34 304 257 27 49 107 113 256 189
  ±3 ±3 ± 4 ± 5 ±11 ± 6 ±29 ±36 ± 1 ± 14 ±10 ± 10 ± 38 ± 19
Conclusions:
Interpretation of results (migrated information):
negative

It can be concluded that neither AHTN itself nor any metabolite generated by the metabolic activation system is mutagenic in the Ames test under the described experimental conditions.
Executive summary:

AHTN was investigated for mutagenic potential using two versions of the Ames test: the standard plate incorporation and the preincubation methods. Seven tester strains were employed (S. typhimurium TA1535, TA1537, TA97, TA98, TA100, TA1O2 and E. coli VP2 uvrA). The cells were exposed to the test compound in absence and presence of a metabolic activation systent (S9 mix) prepared from the livers of phenobarbital/8-naphthoflavone treated rats.

Responsiveness of the strains and activity of the S9 mix were verified by including appropriate positive control compounds.

The test compound was dissolved in DMSO. Milky suspensions concentrations of 158 pg/plate and above. The dose range was chosen 5000 µg/plate. No toxicity was observed up to 5000 µg/plate.

No increase of the number of reverting colonies were observed for any of the seven tester stains.

Thus it can be concluded that neither AHTN nor any of its metabolites are mutagenic in the Ames test under the described conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 Jul 2017 to 21 Aug 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Version / remarks:
adopted 29 July 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material:0000116729
- Expiration date of thebatch: 12 February 2019
- Purity test date: 12 February 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition and physical state of test material: Room temperature solid
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test DMSO was used as solvent (1% DMSO). Different test item stock solutions were prepared and added to the samples.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The solvent was compatible with the survival of the cells and the S9 activity.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solution was treated with ultrasound for 10 minutes at 37 °C and diluted prior to treatment.
Target gene:
Thymidine Kinase gene
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years. These cells are characterised by their high proliferation rate (10-12 h doubling time of the Eurofins Munich stock cultures) and their cloning efficiency, usually more than 50%. The cells obtain a near diploid karyotype (40 ± 2 chromosomes). They are heterozygous at the Thymidine Kinase (TK) locus in order to detect mutation events at the TK- locus. Each cell batch is routinely checked for mycoplasma infection.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: liver microsome preparations (S9mix)
- source of S9 : Eurofins Munich
- method of preparation of S9 mix : Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route. The preparation was performed according to Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
- quality controls of S9: The following quality control determinations were performed: a) Biological activity in: the Salmonella typhimurium assay using 2-aminoanthracene, the mouse lymphoma assay using benzo[a]pyrene, the chromosome aberration assay using cyclophosphamide. b) Sterility Test: A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at ≤ -75°C. The protein concentration in the S9 preparation was 34.2 mg/mL.
Test concentrations with justification for top dose:
- According to OECD Guidelines at least 4 concentrations of the test item were set up in the experiments without and with metabolic activation.The test item was investigated at the following concentrations:
- without metabolic activation: 15, 20, 25, 30, 32, 35 and 38 μg/mL
- with metabolic activation: 10, 15, 20, 25, 30, 35 and 40 μg/mL.
- Justification: The toxicity of the "Any other information on materials and methods including tables" test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Six concentrations [10, 50, 150, 500, 1000 and 2000 μg/mL] were tested without and with metabolic activation. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. 38 μg/mL (without metabolic activation) and 40 μg/mL (with metabolic activation) were selected as the highest concentrations. The experiment without and with metabolic activation was performed as 4 h short-term exposure assay.
Vehicle / solvent:
1% DMSO v/v
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
NUMBER OF REPLICATES:
Duplicates

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4-hour exposure groups both in the absence and presence of metabolic activation. 24-hour exposure group in the absence of metabolic activation

FOR GENE MUTATION:
- Expression time: 6 days
- Selection time: 12 days
- Method used: 96-well plates for the mouse lymphoma assay.
- Selective agent (mutation assays): trifluorothymidine (TFT), Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 μL selective medium with TFT (RPMI 1640 medium supplemented with 20% horse serum, 100 U/100 μg/mL penicillin/streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 25 mM HEPES, 2.5 μg/mL amphotericin B, 5 μg/mL TFT). The plates were scored after an incubation period of about 12 days at 37°C in 5% CO2/95% humidified air.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For a short-term exposure experiment 1 x 10^7 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h the test item was removed by centrifugation (200 x g, 7 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period of 2 days in total at 37 °C in 5% CO2/95% humidified air. The cell density was determined each day and adjusted to 3 x 10^5 cells/mL in a total culture volume of 20 mL, if necessary. After the expression period the cloning efficiency (CE) of the cells was determined by seeding a statistical number of 1.6 cells/well in two 96-well plates. The cells were incubated for at least 6 days at 37 °C in a humidified atmosphere with 5% CO2. Analysis of the results was based on the number of cultures with cell growth (positive wells) and those without cell growth (negative wells) compared to the total number of cultures seeded. Additionally, cultures were seeded in selective medium. Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 μL selective medium with TFT. The plates were scored after an incubation period of about 12 days at 37 °C in 5% CO2/95% humidified air. The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT. For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection. Based on the null hypothesis of the Poisson distribution, the probable number of clones/well (P) is equal to –ln(negative wells/total wells) and the plating efficiency (PE) equals P/(number of cells plated per well). Mutant frequency then was calculated as MF = (PE(cultures in selective medium)/PE(cultures in non-selective medium)). The mutant frequency is usually expressed as “mutants per 10^6 viable cells”.
- Criteria for small (slow growing) and large (fast growing) colonies: Small colonies approximately ≤ ¼ of well diameter, Large colonies approximately > ¼ of well diameter. Size is the key factor and morphology should be secondary

METHODS FOR MEASUREMENT OF CYTOTOXICITY
The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.
Evaluation criteria:
Evaluations of results:
The test item is considered mutagenic if the following criteria are met:
a) The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 10^6 cells and;
b) a dose-dependent increase in mutant frequency is detected. Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations. According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the test result. A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.

Acceptability of the assay:
A mutation assay is considered acceptable if it meets the criteria mentioned in current international guidelines and the current recommendations of the IWGT:
-At least three out of four 96-well plates from the TFT resistance-testing portion of the experiment are scorable
-The cloning efficiency of the negative and/or solvent controls is in the range 65% -120%.
-The spontaneous mutant frequency in the negative and/or solvent controls is in the range 50-170 mutants per 10^6 cells
-The cell number of the negative/solvent controls should undergo 8-32 fold increase during a 2 day growth period (short-term treatment)
-The clastogenic positive controls (MMS and B[a]P) have to produce an induced mutant frequency (total mutant frequency minus concurrent negative control mutant frequency) of at least 300 mutants per 106 cells with at least 40% of the colonies being small colonies or with an induced small colony mutant frequency of at least 150 mutants per 10^6 cells. The RTG must be greater than 10%.
Statistics:
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
No precipitation of the test item was noted in the pre-experiment / main experiment. The relative total growth (RTG) was 16.3% (without metabolic activation) and 16.6% (with metabolic activation) for the highest concentration evaluated.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST SPECIFIC CONFOUNDING FACTORS:
-Effects of pH: No
-Effects of osmolarity: No
-Percipitation: No percipitate was observed during the main test

RANGE-FINDING/SCREENING STUDIES:
-The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 2 mg/mL. Six concentrations [10, 50, 150, 500, 1000 and 2000 μg/mL] were tested without and with metabolic activation. Dose groups that were not evaluable due to very high toxicity of the test item are not reported. The experimental conditions in these pre-experiment were the same as described below in the paragraph experimental performance. After a 2-day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector 1975.

TOXICITY:
-Growth inhibition was observed in the experiment without and with metabolic activation: The relative total growth (RTG) was 16.3% (without metabolic activation) and 16.6% (with metabolic activation) for the highest concentration evaluated.

MUTAGENICITY:
-The mutant frequencies obtained from all experiments were compared with the Global Evaluation Factor (GEF) and a statistical analysis was performed. The GEF is defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data are gathered from ten laboratories. For the microwell method the GEF was defined to be 126. Criterion for mutagenicity is the extension of the GEF by the induced mutant frequency as well as a dose-dependent increase in mutant frequency. The positive controls EMS, MMS and B[a]P showed distinct effects in mutation frequency, thus proving the ability of the test system to detect potential mutagenic effects.
In the main experiment without and with metabolic activation all validity criteria were met. The negative and solvent controls showed mutant frequencies within the acceptance range of 50-170 mutants/10^6 cells, according to the IWGT criteria.
The mutant frequencies induced by the test item did not show any biologically relevant increase. Without metabolic activation the GEF was not exceeded in any dose groups. With metabolic activation the GEF of 126 was exceeded at the concentration of 40 μg/mL (278.3 mutants/10^6 cells), though with a RTG of 16.6%. Whereas the next lower tested concentration of 35 μg/mL Tonalid showed with a RTG of 26.9% a total inconspicuous GEF (19.5 mutants/10^6 cells). According to the OECD guideline positive results with a RTG between 10 and 20% should be interpreted with caution. Whereas based on ICH S2(R1) an effect observed only at the most toxic concentration (RTG reduction ≥ 80%), the weight of evidence indicated a lack of genotoxic potential [7]. In this case the supposed positive result for the highest concentration of 40 μg/mL Tonalid can be considered to be negative. On sponsors request an additional experiment is regarded as not required, due to the narrow range of tested concentrations and the fact that the treatment concentrations meet the acceptance criteria.
A statistical analysis displayed that one of the mutant frequencies was significantly increased over those of the solvent controls, but there was no evidence for a dose-response relationship. Therefore this effect was considered as not biologically relevant.
Historical data for mutant frequencies are shown in the field 'Any other information on results incl. tables' (Table 1). All mutant frequencies for negative, solvent and positive controls were found within the historical range of the test facility Eurofins Munich.

CLASTOGENICITY:
-Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. An extension of the GEF by the induced mutant frequency in combination with an increased occurrence of small colonies (defined by slow growth and/or morphological alteration of the cell clone) is an indication for potential clastogenic effects and/or chromosomal aberrations. The positive controls MMS and B[a]P induced a significant increase in mutant frequency and a biologically significant increase of small colonies (≥40%), thus proving the ability of the test system to indicate potential clastogenic effects.
In the main experiment without and with metabolic activation the percentage of small colonies in the negative controls, the solvent controls, and in the highest dose groups treated with the test item was found to be lower than 40%. Thus, all dose groups were considered as not clastogenic.

A table summarizing the results from the main experiment, without and with metabolic activation is presented in the field 'Any other information on results incl. tables' (Table 2).

Table 1. Historical Laboratory Control Data

(January 2011 to December 2016)

 

NC

PC

S

-S9

+S9

EMS

(300 µg/mL)

MMS

(10 µg/mL)

B[a]P

(2.5 µg/mL)

DMSO

-S9

+S9

Mean

87.9

85.1

726.5

763.4

635.9

90.6

87.0

Min

50.1

50.1

318.7

376.4

303.6

45.6

50.6

Max

170.3

165.9

2919.0

2416.1

1267.2

167.4

159.7

SD

25.5

24.3

203.5

421.6

167.8

30.1

24.8

RSD [%]

29.0

28.6

28.0

55.2

26.4

33.2

28.5

n =

447

653

211

254

255

70

70

NC: Negative Control

S: Solvent Control (1% DMSO)

PC: Positive Controls (+S9 B[a]P, -S9 EMS, MMS)

S9: metabolic activation

Mean: mean of mutant frequency [mutants/ 106 cells]

Min.: minimum of mutant frequency [mutants/ 106 cells]

Max.: maximum of mutant frequency [mutants/ 106 cells]

SD: Standard Deviation

RSD: Relative Standard Deviation

n: Number of control values

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

B[a]P: Benzo[a]pyrene

Table 2 : Summary: Main Experiment, without andwith metabolic activation

 

 

Test Group

 

Conc. [µg/mL]

 

RCEa[%]

 

RTGb[%]

 

MFc[mutants/ 106cells]

 

IMFd

[mutants/ 106cells]

 

GEFe

exceeded

 

Statistical Significant Increasef

 

Precipitate

 

Exp without S9

C1

 

0

102.9

114.4

99.2

/

/

/

-

C2

119.9

136.3

/

/

/

-

S1

0

100.0

100.0

128.3

/

/

/

-

S2

/

/

/

-

3

15

108.1

117.5

112.4

-15.9

-

-

-

4

20

99.7

104.8

136.7

8.3

-

-

-

5

25

113.7

101.6

90.6

-37.7

-

-

-

6

30

92.3

64.7

136.9

8.5

-

-

-

7

32

131.7

56.1

97.0

-31.3

-

-

-

8

35

122.1

36.6

137.1

8.7

-

-

-

9

38

134.3

16.3

207.1

78.7

-

+

-

EMS

300

93.7

98.2

787.4

659.0

+

+

-

MMS

10

69.9

68.8

608.7

480.4

+

+

-

 

 

Exp with S9

C1

0

119.8

118.7

71.4

/

/

/

-

C2

91.5

97.1

/

/

/

-

S1

0

100.0

100.0

76.3

/

/

/

-

S2

/

/

/

-

2

10

124.1

128.6

53.5

-22.8

-

-

-

3

15

113.9

82.1

90.7

14.4

-

-

-

4

20

115.8

63.1

66.4

-9.9

-

-

-

5

25

94.3

37.2

68.7

-7.7

-

-

-

6

30

119.8

41.1

52.9

-23.4

-

-

-

7

35

101.8

26.9

95.9

19.5

-

-

-

8

40

101.8

16.6

354.6

278.3

+

+

-

B[a]P

2.5

98.7

76.8

592.5

516.2

+

+

-

C: Negative Controls

S: Solvent Controls (1% DMSO)

a: Relative Cloning Efficiency, RCE = [(CEdosegroup/ CEofcorrespondingcontrols) x 100] Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100 c: Mutant Frequency,

MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800 d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to solvent controls (Mann Whitney test , p<0.05).

+: significant; -not significant EMS: Ethylmethanesulfonate MMS: Methylmethanesulfonate B[a]P: Benzo[a]pyrene


 

Conclusions:
In conclusion, in the described OECD 490 mutagenicity test under the experimental conditions reported, the test item is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

In this OECD 490 mutagenicity study performed under GLP, the test item Tonalid was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. The test item was investigated at the following concentrations: a) without metabolic activation: 15, 20, 25, 30, 32, 35 and 38 μg/mL and b) with metabolic activation: 10, 15, 20, 25, 30, 35 and 40 μg/mL.

No precipitation of the test item was noted in the experiment. Growth inhibition was observed in the main experiment without and with metabolic activation: The relative total growth (RTG) was 16.3% (without metabolic activation) and 16.6% (with metabolic activation) for the highest concentration evaluated. No biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was only exceeded by the induced mutant frequency at the highest concentration with metabolic activation and an RTG of 16.6%. This increase was considered as biologically not relevant, due to a lack of dose response relationship. Additionally this effect was only observed at a very toxic concentration (RTG reduction ≥ 80%). Furthermore, colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, in the described OECD 490 mutagenicity test under the experimental conditions reported, the test item Tonalid is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 May 1994 - 23 February 1995
Reliability:
1 (reliable without restriction)
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
equivalent or similar to
Guideline:
EPA OTS 798.5375 (In Vitro Mammalian Chromosome Aberration)
Qualifier:
equivalent or similar to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
K1 cell line
Additional strain / cell type characteristics:
other: not used beyond passage 20
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
5.9, 11.7, and 23.4 µg/ml (20-hr harvest without S9)
5.9, 11.7, and 23.4 µg/ml (44-hr harvest without S9)
5.9, 11.7, and 23.4 µg/ml (4-hr exposure; 20-hr harvest without S9)
17.8, 20, and 25 µg/ml (20-hr harvest with S9)
3.9, 7.8, and 15.6 µg/ml (44-hr harvest with S9)
Vehicle / solvent:
acetone
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
other: 2 µg/ml of MNNG (N-methyl-N’-nitro-N-nitrosoguanidine)
Remarks:
20-hr and 44-hr harvests without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
20-hr and 44-hr harvests with S9

Migrated to IUCLID6: 30 µg/ml
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
5E05 cells/25 cm2 flask were seeded for harvests <24 hours
3E05 cells/25 cm2 flask were seeded for harvests>24 hours
DURATION
- Exposure duration: without S9= exposed for 4, 20, and 44 hours and harvested at 20, 44, and 20 hours, respectively; with S9=exposed for 4 hours and harvested at 20 or 44 hours.
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid 2 hours prior to harvest
STAIN (for cytogenetic assays): 5% Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: min. 200 metaphase spreads
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
OTHER EXAMINATIONS:
- Determination of polyploidy: scored for chromatid-type and chromosome-type aberrations
- Other: XY coordinates for each cell with chromosomal aberrations were recorded
OTHER: The percent polyploidy cells per 100 metaphase cells recorded (delayed harvest). Mitotic index=percentage of cells in mitosis per 500 cells counted.
Evaluation criteria:
The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the initial and independent repeat assays. The toxic effects of treatment as measured by cell growth inhibition will also be reported in the confirmatory assay for the 4 hour pulse treatments collected 20 hours after initiation of treatment. The number and types of aberrations found, the percentage of structurally damaged cells (percent aberrant cell) in the total population of cell examined, and the frequency of structural aberrations per cell (mean aberrations per cell) was calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cell with one or more aberrations or in the frequency of structural aberrations per cell.
All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cell with aberrations are increased in a dose responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p ≤ 0.05). A reproducible and significant increase at a single dose level will be considered positive. Test articles not demonstrating a statistically significant increase in aberrations will be considered negative.
Statistics:
Fisher’s exact test (percent aberrant cells); Fisher’s test (pairwise comparison of the percent aberrant cells of each treatment group compared to solvent controls); Cochran-Armitage test (measure dose-responsiveness in positive Fisher’s test).
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
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:
Without S9 (20-hr exposure/20-hr harvest), cells treated with up to 23.4 µg AHTN/ml were scored; cytotoxicity noted at all concentrations. For concentrations of 0 (untreated), 0 (solvent control), 5.9, 11.7, and 23.4 µg/ml, mitotic index was 6.8, 5.9, 5.5, 4.7, and 3.0, respectively; mean (+/-SD) aberrations per cell were 0.025+/-0.157, 0.025+/-0.186, 0.025+/-0.157, 0.020+/-0.172, and 0.005+/-0.071, respectively; and cells with aberrations were 2.5, 2.0, 2.5, 1.5, and 0.5%, respectively.
Without S9 (44-hr exposure/44-hr harvest), cells treated with up to 23.4 µg AHTN/ml were scored; cytotoxicity noted at all concentrations. For concentrations of 0 (untreated), 0 (solvent control), 5.9, 11.7, and 23.4 µg/ml, mitotic index was 7.7, 5.9, 5.7, 5.5, and 2.4, respectively; mean (+/-SD) aberrations per cell were 0.000+/-0.000, 0.050+/-0.707, 0.000+/-0.000, 0.010+/-0.100, and 0.000+/-0.000, respectively; and cells with aberrations were 0.0, 0.5, 0.0, 1.0, and 0.0%, respectively.
Without S9 (4-hr exposure/20-hr harvest), cells treated with up to 23.4 µg AHTN/ml were scored; cytotoxicity noted at all concentrations. For concentrations of 0 (untreated), 0 (solvent control), 5.9, 11.7, and 23.4 µg/ml, mitotic index was 9.9, 10.8, 9.5, 8.3, and 5.8, respectively; mean (+/-SD) aberrations per cell were 0.030+/-0.299, 0.000+/-0.000, 0.010+/-0.100, 0.005+/-0.071, and 0.010+/-0.100, respectively; and cells with aberrations were 1.5, 0.0, 1.0, 0.5, and 1.0%, respectively.
With S9 (4-hr exposure/20-hr harvest), cells treated with up to 25 µg AHTN/ml were scored; cytotoxicity noted at all concentrations. For concentrations of 0 (untreated), 0 (solvent control), 17.8, 20, and 25 µg/ml, mitotic index was 7.1, 8.0, 4.7, 2.3, and 1.6, respectively; mean (+/-SD) aberrations per cell were 0.040+/-0.196, 0.015+/-0.122, 0.115+/-0.427, 0.100+/-0.347, and 0.125+/-0.795, respectively; and cells with aberrations were 4.0, 1.5, 8.5, 8.5, and 5.5%, respectively. All AHTN treated cultures had significantly increased cells with aberrations compared to controls; however, authors concluded that the response of the high concentration was within historical range of untreated control and low and mid concentration increases were only slightly out of the historical range plus no dose-response was evident.
With S9 (4-hr exposure/44-hr harvest), cells treated with up to 15.6 µg AHTN/ml were scored; cytotoxicity noted at all concentrations. For concentrations of 0 (untreated), 0 (solvent control), 3.9, 7.8, and 15.6 µg/ml, mitotic index was 6.5, 6.1, 7.0, 5.6, and 1.7, respectively; mean (+/-SD) aberrations per cell were 0.050+/-0.329, 0.010+/-0.100, 0.020+/-0.140, 0.020+/-0.172, and 0.035+/-0.210, respectively; and cells with aberrations were 3.5, 1.0, 2.0, 1.5, and 3.0%, respectively.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

AHTN was concluded to be negative for chromosome aberrations under the conditions of this study.
Executive summary:

A cytogenetic assay with Chinese Hamster ovary (CHO-K1) cells was conducted with AHTN according to OECD Guideline 473 at concentrations of 5.9, 11.7 or 23.4 μg/ml in the non-activated study, using 4, 20 and 44 hr exposure periods. In the S-9 (from rat liver induced by Aroclor 1254) activated study, dose levels of 17.8, 20 and 25 μg/ml were tested for the 4-hr exposure period with a 20-hr harvest time and at dose levels of 3.9, 7.8 and 15.6 μg/ml for the 4-hr exposure period with a 44-hr harvest time. At the 4, 20 and 44-hr harvest times; the cells were assessed for structural chromosome aberrations, and at the 44-hr harvest time also for numerical chromosome aberrations. N-methyl-N’-nitro-Nnitrosoguanidine was used as a positive control in the non-activated study and benzo(a)pyrene in the activated study.

The mitotic index was significantly lowered at the highest dose in all cases. Positive controls caused increases in structural (significant) and numerical aberrations (significant in the case of B(a)P) in all cases. No significant increases in structural or numerical chromosome aberrations were observed for AHTN without metabolic activation at any dose.

With metabolic activation, AHTN induced statistically significant increases in structural aberrations at all doses at the 20-hr harvest time (not dose-related and with significant cytotoxicity at all doses), as well as a statistically significant increase in numerical aberrations at the highest dose at the 44-hr harvest time. The latter increase was, however, still within historical control range. There was no dose response observed in this portion of the assay. Hence, the authors concluded AHTN to be negative for structural and numerical chromosome aberrations in this test.

Source: EU Risk Assessment Report, European Chemicals Bureau (May 2008)

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

Genetic toxicity in vivo

Description of key information

The test item was not found to be mutagenic in vivo. Under the conditions of the assay described in this report, the test item did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: other: Incidence of micronucleated polychromatic erythrocytes
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study and range finding for OECD method 474 for mouse micro nucleus study. The protocol has been written to comply with EEC Directive 79/831 Annex V, B.12 Mutagenicity (Micronucleus Test), June, 1989; OECD Guideline 474 (Genetic Toxicology: Micronucleus Test); EPA Health Effects Testing Guidelines, Subpart 798.5395 (In Vivo Mammalian Bone Marrow Cytogenetics Tests: Micronucleus assay). Fed. Register. vol. 50. September, 1985 with revisions Fed. Register. vol. 52, May, 1987, and Notification No. t 18 of the Pharmaceutical Affairs Bureau, Ministry of Health and Welfare, Japan, February 15, 1984.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus/cytogenetic assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
ICR mice were obtained from Harlan Sprague Dawley, Inc., Frederick, MD. At the initiation of the study, the mice were 6 to 8 weeks old. Males, 28.1-37.2 grams at randomization Females, 24.5-31.0 grams at randomization.
The mice were housed in an AAALAC-accredited facility with a controlled environment of 20-27°C average 23°C, 50±20% relative humidity, and a 12 hour light/dark cycle. Mice of the same sex were housed up to five per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and were covered with filter material. Heat-treated hardwood chips were used for bedding. Mice had free access to certified laboratory rodent chow which had been analyzed for environmental contaminants (Harlan TEKLAD certified Rodent 7012C) and to tap water (Washington Suburban Sanitary Commission, Potomac Plant). There are no contaminants in the feed which are expected to influence the study. The water meets USEPA drinking water standards and is monitored at least annually for levels of organophosphorus pesticides, metals, and coliform and other contaminants.
Route of administration:
intraperitoneal
Duration of treatment / exposure:
24, 48 and 72 hours (see table 1)
Frequency of treatment:
single treatment
Post exposure period:
72 hours
Remarks:
Doses / Concentrations:
400, 800 and 1600 mg/kg body weight
Basis:
other: Intraperitoneal
No. of animals per sex per dose:
goups of 5 male and 5 female
Control animals:
yes, concurrent vehicle
Positive control(s):
Gyclophosphamide (CP, CAS 6055-19-2), was obtained from Sigma Ghemical Company and was dissolved in sterile distilled water at a concentration of 3 mg/ml.
Tissues and cell types examined:
Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained and permanently mounted.
Details of tissue and slide preparation:
Cells were treated and well spread and stained. 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970; Mackey). The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤0.05, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a Valid Test
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p≤0.05, Kastenbaum-Bowman Tables).
Statistics:
Kastenbaum, M.A. and K.O. Bowman. 1970. Tables for determining the statistical significance of mutation frequencies. Mutation Res. 9:527-549.
Sex:
male/female
Genotoxicity:
negative

Reductions of up to 23% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to their respective vehicle controls. The reduction in the frequency of polychromatic erythrocytes in the bone marrow suggest that there was toxicity and bioavailability of the test article to the bone marrow target tissue.

The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in test article treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (table 2).

Gyclophosphamide induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice.

Conclusions:
Interpretation of results (migrated information): negative
All criteria for a valid test were met. Under the conditions of the assay described in this report, AHTN did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

ICR mice were dosed with 400, 800, or 1600 mg/kg bw AHTN in corn oil by intraperitoneal injection. The high dose was selected to be 80% of the estimated intraperitoneal LD50 . The positive control was cyclophosphamide. Bone marrow was collected at 24, 48 and 72 hr after dosing and examined for micronucleated polychromatic erythrocytes (PCE). No mortality occurred. Clinical signs consisted of lethargy at all dose levels and diarrhoea at 1600 mg/kg bw. Moderate reductions (up to 23%) in the ratio of PCE to total erythrocytes were observed in some treated groups suggesting toxicity and bioavailability to the bone marrow target. The positive control induced a significant increase in micronucleated PCE in both male and female mice at 24 hr (the only harvest time for this group). No significant increase in micronucleated PCE in AHTN-treated groups relative to the respective vehicle control group was observed in male or female mice at 24, 48 or 72 hr after dose administration.

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

Additional information

Short description of key information:

A number of different in-vitro genetic toxicity tests and a in-vivo micronucleus test were performed to demonstrate negative genetic toxicity.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

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