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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test: The study was performed to investigate the potential of KARMALONE to induce gene mutations according to OECD guideline No 471.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, KARMALONE is considered to be non-mutagenic in this Salmonella typhimu­ rium reverse mutation assay.

Chromosome aberration test:

The test item COSMONE, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitro in two independent experiments according to OECD Guideline no. 473. It can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.

Therefore, COSMONE is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic concentrations in the absence and the presence of metabolic activation.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 14 to February 03 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Identity: KARMALONE
Batch No.: TQT0300497
Aggregate State at Room Temperature: liquid
Colour: colourless to pale yellow
Purity: 90.9 %
Stability in Solvent: not indicated by the sponsor
Storage: room temperature
Expiration Date: November 28, 2004
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9
Phenobarbital/ -Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar Hanlbm rats, weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCI solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80° C. Small numbers of the ampoules can be kept at -20°C for up to one week.

The protein concentration in the S9 preparation was 27.2 mg/ml (lot no. R 071103).

Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 15% vlv in the cultures. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix:

8 mM MgCl2
33 mM KCI
5 mM Glucose-6-phosphate
5 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al. .
Test concentrations with justification for top dose:
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Based on the toxic effects observed in strain TA 1537 at 2500 and 5000 µg/plate the same concentration range was chosen for the main experiments.

The concentration range included two logarithmic decades. The following concentrations were tested:
3; 1O; 33, 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO (purity > 99 %, MERCK, D-64293 Darmstadt)
The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
The test item precipitated in the overlay agar at 1000 µg/plate and above in experiment I, and at 333 µg/plate and above in experiment 11.
The undissolved particles of the test item had no influence on the data recording.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine, 4-NOPD and 2-aminoanthracene, 2-AA
Details on test system and experimental conditions:
Pre-experiment:
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre­ experiment were the same as described for the experiment I below (plate incorporation test).

Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>O colonies) at five concentrations or more in all strains used.

Experimental performance:
For each strain and dose level, including the controls three plates were used.

The following materials were mixed in a test tube and poured onto the selective agar plates:
- 100 µLTest solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
- 500 µL S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
- 100 µL Bacteria suspension (cf. test system, pre-culture of the strains),
- 2000 µL Overlay agar

In the pre-incubation assay 100 µL test solution, 500 µL S9 mix/ S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and incubated at 37°C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45° C) was added to each tube. The mixture was poured on minimal agar plates.

After solidification the plates were incubated upside down for at least 48 hours at 37° C in the dark.
Rationale for test conditions:
In accordance with the relevant guidelines.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration .
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not required.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Reduced background growth was observed in the presence of metabolic activation from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment II.

Toxic effects; evident as a reduction in the number of revertants were observed at the following concentrations (µg/plate) (see table 1)
No toxic effects observed

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with KARMALONE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

The historical range of positive controls was exceeded in strains TA 1535 and TA 100 without metabolic activation in experiments I and II. This effect indicates the sensitivity of the strains rather than compromising the assay.

In experiment II, the data in the negative of strain TA 98 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct in­ crease in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Table 1:

Strain

Experiment I

Experiment11

 

without S9 mix

with S9 mix

without S9 mix

with S9 mix

TA 1535

I

1000, 5000

I

333-5000     I

TA 1537

I

2500, 5000

I

2500,5000

TA98

I

I

I

I

TA 100

I

I

I

I

TA 102

I

I

I

5000

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, KARMALONE is considered to be non-mutagenic in this Salmonella typhimu­ rium reverse mutation assay.
Executive summary:

This study was performed to investigate the potential of KARMALONE to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA102.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 3; 1O;33, 100; 333; 1000; 2500; and 5000 µg/plate

 

Reduced background growth was observed in the presence of metabolic activation from 1000 to 5000 µg/plate in experiment I and from 333 to 5000 µg/plate in experiment11.

 

In the presence of metabolic activation, toxic effects, evident as a reduction in the number of revertants, were observed in strains TA 1535 and TA 1537 in experiment I and II, and in strainTA102inexperiment11.

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with KARMALONE at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls and showed a distinct in­ crease of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

Therefore, KARMALONE is considered to be non-mutagenic in this Salmonella typhimu­ rium reverse mutationassay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 30 2005 to December 14 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
conducted under GLP conditions
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Identity: COSMONE
Batch No.: 9000607060
Aggregate State at Room Temperature: Liquid
Colour: Colourless to pale yellow
Molecular Weight: 222 g/mol
Purity: 90.6 %
Solubility in Water: 4.6 mg/L
Stability in Solvent: Not indicated by the sponsor
Storage: At room temperature, protected from light
Expiration Date: May 17, 2007
Target gene:
not applicable (chromosome aberration test)
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Large stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt, D-64287 Darmstadt) were stored in liquid nitrogen in the cell bank of RCC Cytotest Cell Research GmbH allowing the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasm contamination and checked for karyotype stability. Consequently, the parameters of the experiments remain similar because of standardized characteristics of the cells.

Thawed stock cultures were propagated at 37° C in 80 cm² plastic flasks (GREINER, D-72632 Frickenhausen). About 5 x 105 cells per flask were seeded into 15 mL of MEM (Minimal Essential Medium; SEROMED; D-12247 Berlin) supplemented with 10 % fetal calf serum (FCS; PAA Laboratories GmbH, D-35091 Cölbe). The cells were subcultured twice weekly. The cell cultures were incubated at 37° C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Phenobarbital/-Naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats, weight approx. 220 - 320 g (supplied from RCC Ltd; Laboratory Animal Services, CH- 4414 Füllinsdorf) induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D- 22335 Hamburg) and -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1:3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at -80° C. Small numbers of the ampoules were kept at -20°C for up to one week.

The protein concentration was 36.4 mg/mL (Lot no. 240605) in the pre-experiment and Experiment I and II.

An appropriate quantity of 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 following concentrations:

8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP

in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.

During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al..
Test concentrations with justification for top dose:
The highest concentration used in the cytogenetic experiments was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible.
With respect to the molecular weight and the purity of the test item, 2450 µg/mL of COSMONE (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 19.1 and 2450 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity.
Using reduced cell numbers as an indicator for toxicity in the pre-test, clear toxic effects were observed after treatment with 19.1 µg/mL and above in the absence of S9 mix and with
306.3 µg/mL and above in the presence of S9 mix. Considering the toxicity data and the occurrence of test item precipitation in the pre-test, 40 µg/mL (without S9 mix) and 400 µg/mL (with S9 mix) were chosen as top concentrations in Experiment I.
Dose selection of Experiment II was also influenced by test item toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 19.1 µg/mL and above. Therefore, 40 µg/mL (28 hrs preparation interval) and 80 µg/mL (18 hrs preparation interval) were chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 400 µg/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.
Vehicle / solvent:
On the day of the experiment (immediately before treatment), the test item was dissolved in ethanol (E. MERCK, D-64293 Darmstadt; purity 99.8 %). The final concentration of ethanol in the culture medium was 0.5 % (v/v). The solvent was chosen to its solubility properties and its relative nontoxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Treatment:
Exposure period 4 hours:
The culture medium of exponentially growing cell cultures was replaced with serum-free medium (for treatment with S9 mix) or complete medium (for treatment without S9 mix) with 10 % FCS (v/v), containing the test item. For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 hrs the cultures were washed twice with "Saline G" and then the cells were cultured in complete medium for the remaining culture time.

The "Saline G" solution was composed as follows (per litre):
NaCl 8000 mg
KCl 400 mg
Glucose x H20 1100 mg Na2HP04 x 7H20 290 mg KH2P04 150 mg

pH was adjusted to 7.2

Exposure period 18 and 28 hours:
The culture medium of exponentially growing cell cultures was replaced with complete medium (with 10 % FCS) containing different concentrations of the test item without S9 mix. The medium was not changed until preparation of the cells.
All cultures were incubated at 37° C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Preparation of the Cultures:
Colcemid was added (0.2 µg/mL culture medium) to the cultures 15.5 hrs and 25.5 hrs, respectively after the start of the treatment. The cells on the slides were treated 2.5 hrs later, in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37° C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). Per experiment two slides per group were prepared. After preparation the cells were stained with Giemsa (E. Merck, D-64293 Darmstadt).
Evaluation criteria:
A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and/or
- no significant increase of the number of structural chromosome aberrations is observed. A test item is classified as clastogenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, exclusive gaps).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher´s exact test (9) (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criteria is valid:
A test item can be classified as aneugenic if:
− the number of induced numerical aberrations is not in the range of our historical control data (0.0 - 8.5 % polyploid cells).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In Experiment I and II, in the presence of S9 mix, precipitation of the test item in culture medium was observed after 4 hrs treatment with 200 µg/mL and above.

In Experiment I in the absence of S9 mix, and in Experiment II in the presence of S9 mix, no clear toxic effects indicated by reduced cell numbers (see Table 5, page 25) and/or mitotic indices (see Table 6 and 9, page 26 and 29) of below 50 % of control were observed up to the highest scorable test item concentration. However, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
In contrast, the mitotic indices were strongly reduced after 18 hrs continuous treatment with 20 µg/mL (36.5 % of control) in Experiment II in the absence of S9 mix and after 4 hrs treatment with 100 µg/mL (32.9 % of control) in Experiment I at 18 hrs preparation interval in the presence of S9 mix. In addition, the cell numbers were clearly reduced after 28 hrs continuous treatment with 20 µg/mL (48.8 % of control) in Experiment II in the absence of S9 mix and after 4 hrs treatment with 100 µg/mL (48.9 % of control) in Experiment I at 18 hrs preparation interval in the presence of S9 mix.

In both cytogenetic experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed (see Table 7 – 8 and Table 10 – 11, page 27, 28, 30, and 31). The aberration rates of the cells after treatment with the test item (0.0 - 3.0 % aberrant cells, exclusive gaps) were close to the range of the solvent control values (0.5 - 2.5 % aberrant cells, exclusive gaps) and within the range of our historical control data: 0.0 - 4.0 % aberrant cells, exclusive gaps. A single statistical significant (p < 0.05; page 32) increase was observed in Experiment II, in the presence of S9 mix, at 28 hrs preparation interval, after treatment with 200 µg/mL. Although this increase of 3.0 % aberrant cells was statistically significant compared to the low response (0.5 % aberrant cells) in the solvent control data, the response is within the historical control data range (0.0 - 4.0 % aberrant cells, exclusive gaps).
In addition, dose-related increases in the number of aberrant cells were observed at 5 to 20 µg/mL in Experiment II, after 18 hrs continuous treatment in the absence of S9 mix (0.5 %, 2.0 %, and 2.5 % aberrant cells) and at 25 to 100 µg/mL in Experiment I, in the presence of S9 mix at 18 hrs preparation interval (0.0 %, 1.5 %, and 2.0 % aberrant cells). Also these values were clearly within the range of our laboratory’s historical control data. Therefore, the statistical significance and the dose-dependencies have to be regarded as being biologically irrelevant.

Table 6 and 9, page 26 and 29; show the occurrence of polyploid metaphases. In both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (1.1 - 2.3 %) as compared to the rates of the solvent controls (0.9 - 2.1 %).

In both experiments, EMS (300 and 400 µg/mL, respectively) and CPA (1.4 and 2.0 µg/mL, respectively) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item COSMONE did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) when tested up to cytotoxic concentrations in the absence and presence of metabolic activation.
Conclusions:
In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, COSMONE is considered to be non-clastogenic in this chromosome aberration test when tested up to cytotoxic concentrations in the absence and the presence of metabolic activation.
Executive summary:

The test item COSMONE, dissolved in ethanol, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamsterin vitroin two independent experiments. The following study design was performed:

 

Without S9 mix

With S9 mix

 

Exp. I

Exp. II

Exp. I

Exp. II

Exposure period

4 hrs

18 hrs

28 hrs

4 hrs

4 hrs

Recovery

14 hrs

-

-

14 hrs

24 hrs

Preparation interval

18 hrs

18 hrs

28 hrs

18 hrs

28 hrs

In each experimental group two parallel cultures were set up. Per culture 100 metaphase plates were scored for structural chromosome aberrations.

The highest applied concentration in the pre-test on toxicity (2450 µg/mL; approx. 10 mM) was chosen with regard to the molecular weight and the purity of the test item with respect to the current OECD Guideline 473.

Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation. The chosen treatment concentrations are described in Table 2 (page 16). The evaluated experimental points and the results are summarised in Table 1 (page 10).

In this study, clear cytotoxicity was observed after test item treatment. However, in Experiment I in the absence of S9 mix and in Experiment II in the presence of S9 mix concentrations showing clear toxic effects were not scorable for cytogenetic damage.

In both independent experiments, no biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item. The statistical significance observed in Experiment II in the presence of S9 mix, and the dose-dependencies observed in Experiment I in the presence of S9 mix and in Experiment II after 18 hrs treatment in the absence of S9 mix have to be regarded as biologically irrelevant, because all values were clearly within the range of our laboratory’s historical control data (0.0– 4.0 % aberrant cells exclusive gaps)

No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

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

Additional information

Justification for classification or non-classification

Based on the study results which are negative, Cosmone should not be classified as mutagenic nor clastogenic according to the EU CLP regulation (No 1272/2008 and its adaption 286/2011).