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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The following in vitro studies, all performed with and without metabolic activation, all showed negative results:

- Salmonella typhimurium reverse mutation assay (TA1535,TA1537,TA98 and TA100), OECD 471

- V79-HGPRT (V79), OECD 476

- Chromosome abberation test (CHO cells), OECD 473
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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted reliable study according to current guidelines.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
less strains
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced SD rat S9mix
Test concentrations with justification for top dose:
5000, 1000, 200, 40, 8 µg/plate
Due to the substance's toxicity, doses ranging from 25 µg to 800 µg per plate were chosen for the repeat tests:
800, 400, 200, 100, 50, 25 µg/plate
Vehicle / solvent:
The solvent employed for Hallcomid M-8-10 was ethanol and for the positive controls DMSO.
No "untreated" negative control was set up for ethanol, since sufficient evidence was available in the literature
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Remarks:
No "untreated" negative control was set up for ethanol, since sufficient evidence was available in the literature
Positive controls:
yes
Positive control substance:
other: Sodium azide for TA 1535; Nitrofurantoin for TA 100; 4-nitro-1,2-phenylene diamine for TA 1537 and TA 98; 2-aminoanthracene for all strains (with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration:48h
- Selection time (if incubation with a selection agent):48h

NUMBER OF REPLICATIONS: performed in quadruplicates (two experiments)

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method:The toxicity of the substance was assessed in three ways: Background growth, by marked and dose-dependent reduction in the mutant count per
plate compared to the negative control and bacterial counts were taken on two plates for each concentration studied with S9 mix. However,
if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and
the laboratories' own historical data (see Chapter 8).
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience (see Chapter 8).
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.
An assay which did not comply with at least one of the above criteria was not used for assessment.

Assessment Criteria
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at
least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines
may be overruled by good scientific judgement.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
but tested up to the limit, reduced concentrations in second experiment
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
As may be seen, there was no indication of a bacteriotoxic effect of Hallcomid M-8-10 at doses of up to and including 100 µg per plate. The total
bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore, they could only be used to a limited extent up to 1000 µg per plate for assessment purposes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls.The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased mutant

counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the

S9 mix.

Conclusions:
Interpretation of results (migrated information):
negative

The registered substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test.
Executive summary:

The registered substance was investigated using the Salmonella/microsome test for point mutagenic effects in doses of up to

5000 µg per plate on four Salmonella typhimurium LT2 mutants according to OECD 471. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

Doses of up to and including 100 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged

and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic

effect, so that this range could only be used to a limited extent up to 1000 µg per plate for assessment purposes.

Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count,

in comparison with the negative controls, was observed.

Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked

mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative

controls.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to guideline, well conducted (GLP) and documented
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
in vitro cytogenetic test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from Aroclor 1254 treated wistar rats
Test concentrations with justification for top dose:
0, 10, 40, 160 µg/ml (without S9)
0, 7.2, 36, 180 µg/ml (with S9)
with harvest time 24h
highest concentration again additional with harvest time 8h and 30 h tested
Pretest with concentration up to 1000 µg/ml showed less survived cells in concentration of 160 µg/ml and above
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol (stock solution containing 500mg/ml in ethanol was diluted in culture medium up to a concentration of 1mg/ml
- Justification for choice of solvent/vehicle: solubility
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Remarks:
untreated control
Positive controls:
yes
Positive control substance:
other: mitomycin C and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): 8, 24h (main study), 30h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): 5% Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: 1 x 10e6 cells were seeded in 20ml medium (containing 0.2ml testitem in solvent, and 1mL S9 mix if metabolic activation)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index and cell survival
Evaluation criteria:
The mitotic index was determined by counting 1000 cells per culture.
Chromosomes of approximately 200 metaphases per concentration, 100 metaphases from each of two parallel cultures, were
examined for structural changes.
classes of aberrations are characterized as follows:
Gaps
Break
Fragment
Deletion
Exchange
Multiple abberation
Statistics:
The statistical analysis of the results was performed by pair-wise comparison of the numbers of
metaphases with aberrations (including and excluding gaps) and of metaphases with exchanges among 200 cells for treatment
and solvent control groups.
The statistical analysis followed the recommendations outlined by Richardson et al. (1989)
Fisher exact test was used for the statistical evaluation.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
tested up to first cytotoxic concentration 160µg/ml 180µg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: 2 pre studies - first pre study ranging from 10 to 1000µg/ml
- secound pre study ranging from 100 to 250 µg/ml

COMPARISON WITH HISTORICAL CONTROL DATA: historical control data were added to the study report.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Survival index determined, highest tested concentration reduces the index by a minimum of 40% (43.2 ; 160µg/ml without S9 and 66.7%; 180µg/ml with S9) harvest time of 8 h, only moderate to negligible effects (with no effects) were seen for harvest times of 24h and 30h
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

Not considered to be clastogenic for mammalian cells with and without metabolic activation
Executive summary:

Chinese hamster ovary cells were treated with the registered substance at the concentrations of 10, 40 and 160 µg/ml medium without S9 mix and at the concentrations of 7.2, 36 and 180 µg/ml with S9 mix. Th tes substance induced cytotoxic effects in cells exposed to the highest doses with and without metabolic activation at the early harvest time of 8 hours after the beginning of the treatment. The mitotic indices were markedly reduced to 66.7 and 43.2 % relative to control cells.

With one exception, no statistically significant or biologically relevant increases of numbers of metaphases with aberrations were detected 8, 24 or 30 hours after the beginning of the four hour treatment with the test substance with and without S9 mix (Tables 2-5). A statistically significant increase of the numbers of cells with aberrations was calculated for cells exposed to the dose of 180 µg/ml with metabolic activation and the harvest time of 8 hours. However, the absolute number of cells with aberrations (3.5 %) was within the normal range as compared to the values for other solvent controls within this study and for other studies performed in this laboratory. This statistically significant increase was therefore considered to be caused by the unusually low number of cells with aberrations in the corresponding solvent control. This statistically significant result is therefore considered not to be biologically relevant.

The positive controls mitomycin C and cyclophosphamide induced clear clastogenic effects and demonstrated the sensitivity of the test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to guideline, well conducted (GLP) and documented
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 cell line was originally derived from the lung of a
male Chinese hamster (Chu and Mailing, 1968) kind gift from Prof. G. Speit, University of Ulm, Germany
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix Aroclor 1254 induced from Wistar rats
Test concentrations with justification for top dose:
Cytotoxicity test: 7.9, 15.7, 31.3, 62.5, 125.0, 250.0, 500.0, 750.0, 1000.0 µg/ml
Main test: 25.0, 50.0, 100.0, 125.0, 150.0, 200.0, 250.0µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulfonate (EMS) and Dimethylbenzanthracene (DMBA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium (containing 2% FCS)

according to Guideline

DURATION
- Preincubation period: 16-24h
- Exposure duration: 5h
- expression period: 6 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-Thioguanine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: triplicates for cytotoxicity
Main test (repeated completly two times ): 8 dishes (from 1 flask) were examined, for each dosage 2 flask were used.

NUMBER OF CELLS EVALUATED:
for each dose level one flask with 4*10e6 cells were avaiable.

DETERMINATION OF CYTOTOXICITY
- Method: cell count, average count, cytotoxicity was expressed by comparison of
colonies in treated cultures versus vehicle control cultures (relative cloning efficiency)

Evaluation criteria:
The number of 6-TG resistant colonies in the mutation assay dishes and the number of colonies in the
cloning efficiency dishes were determined.Colonies with 50 cells or less were excluded
Statistics:
Weighted analysis of variance as well as to a weighted recursive regression, both with Poisson derived weights. Weighted analysis of
variance followed by pairwise comparisons using Dunnetts test.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
up to cytotoxic concentration
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Precipitation of the test article after addition of the test article-Ethanol solution to
the medium starting to be evident at 1500 µg/ml. Therefore, a preliminary cytotoxicity test was conducted with a series of
9 concentrations ranging from 7.9 µg/ml to 1000 µg/ml
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The registered substanceis considered to be nonmutagenic in the V79-HGPRT Forward Mutation Assay both with and without metabolic activation.
Executive summary:

The registered substance was assayed for mutagenic activity at the HGPRT locus in V79 cells from 25 to 250 µg/ml both, with and without metabolic activation according to OECD guideline 476. Under both treatment conditions, cytotoxic effects were induced . The vehicle control mutant frequencies were all in the normal range of background frequencies for the assay. In contrast, the positive controls EMS and DMBA induced a distinct mutagenic effect in mutant frequency, which was significantly increased over the negative controls demonstrating the sensitivity of the test system and the ability to detect known mutagens.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Valid in vitro data to assess the genetic toxicity of the registered substance are available.

The registered substance was investigated using the Salmonella/microsome test for point mutagenic effects in doses of up to 5000 µg per plate on four Salmonella typhimurium LT2 mutants according to OECD 471 (Bayer 1992, B. A. Herbold). These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98. Doses of up to and including 100 µg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to 1000 µg per plate for assessment purposes. Evidence of mutagenic activity was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. Therefore, the registered substance was considered to be non-mutagenic without and with S9 mix in the Salmonella/microsome test.

A further study was carried out to observe the genotoxic potential the registered substance(Bayer, 1994, S. Brendler-Schwaab). Therefore the test material was assayed for mutagenic activity at the HGPRT locus in V79 cells from 25 to 250 µg/ml both, with and without metabolic activation according to OECD guideline 476. Under both treatment conditions, cytotoxic effects were induced. The vehicle control mutant frequencies were all in the normal range of background frequencies for the assay. In contrast, the positive controls and DMBA induced a distinct mutagenic effect in mutant frequency, which was significantly increased over the negative controls demonstrating the sensitivity of the test system and the ability to detect known mutagens. Following the test substance was considered to be non mutagenic in the V79-HGPRT Forward Mutation Assay both with and without metabolic activation.

Additionally, the genotoxic potential of the registered substance was investigated in an in vitro mammalian chromosome abberation test according to OECD 473 (Bayer 1995; R. Gahlmann). Therefore Chinese hamster ovary cells were treated with the substance at the concentrations of 10, 40 and 160 µg/ml medium without S9 mix and at the concentrations of 7.2, 36 and 180 µg/ml with S9 mix. The test substance induced cytotoxic effects in cells exposed to the highest doses with and without metabolic activation at the early harvest time of 8 hours after the beginning of the treatment. The mitotic indices were markedly reduced to 66.7 and 43.2 % relative to control cells. With one exception (which was considered as uncritical, see study summary), no statistically significant or biologically relevant increases of numbers of metaphases with aberrations were detected 8, 24 or 30 hours after the beginning of the four hour treatment with the test substance with and without S9 mix. Therefore, the test substance was not considered to be clastogenic for mammalian cells with and without metabolic activation

 

Assessment of genetic toxicity

In summary there were no positive findings for gene mutation or cytogenicity from in vitro genotoxicity test performed with the registered substance.

 

Following the mutagenicity testing strategy no in vivo experiment is proposed due to this result.

Justification for selection of genetic toxicity endpoint

For each of the three required in vitro genotoxicity study, a relevant and reliable study is available.

Short description of key information:

- Salmonella typhimurium reverse mutation assay (TA1535,TA1537,TA98 and TA100), OECD 471: non-mutagenic (with and without metabolic activation) (Bayer 1992, B. A. Herbold)

- V79-HGPRT (V79), OECD 476: non mutagenic (with and without metabolic activation) (Bayer 1994, S. Brendler-Schwaab)

- Chromosome abberation test (CHO cells), OECD 473: not clastogen (with and without metabolic activation) (Bayer 1995; R. Gahlmann)

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In vitro genotoxicity test and in vitro mutagenicity test does not reveal a positive result.

Due to criteria of GHS (Regulation (EU) 1272/2008) for germ cell mutagens ("The classification in Category 2 is based on: positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from: somatic cell mutagenicity tests in vivo, in mammals; or other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays....") the substance is not to classify as germ cell mutagen. Also according to EU-criteria DSD (67/548/EEC) the available information does not lead to a classification.

Labelling genotoxicity/mutagenicity:

GHS: no classification

DSD: no classification