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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three in-vitro tests (2 Ames tests and a Chromosome Aberration test) are available.

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: 92/69/EWG, B.14 (Ames-Test)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation system:
S9-Mix mit 10 % S9 Fraktion von Aroclor induzierten männlichen Sprague Dawley Ratten "ENGLISH" S9 mix with 10 % S9 fraction of Aroclor 1254 induced male Sprague Dawley rats.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50, 158, 500, 1581, 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50, 158, 500, 1581, 5000 µg/plate
Vehicle / solvent:
Solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Na-azide, NF, 4-NPDA, Cumene, 2-AA
Details on test system and experimental conditions:
Concentration of the test substance resulting in precipitation: 1581 µg/plate and higher.
Evaluation criteria:
Postive controls: sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide, 2-aminoanthracene increased mutant counts to well over those of the negative control, and thus demonstrated the system's sensitivity and the activity of the S9 mix
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: None


"ENGLISH"

none

Vulcuren VP KA 9188 was considered to be non-mutagenic with and without metabolic activation in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

Precipitation of the test substance: 1581 µg/plate and higher.

Conclusions:
Interpretation of results: negative with and without metabolic activation.
Executive summary:

Vulcuren VP KA 9188 was considered to be non-mutagenic with and without metabolic activation in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. No bacteriotoxic effects were seen in any of the dose groups evaluated (Bayer 2000a).

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
other: 92/69/EWG, B.10 (Säuger zytogenetischer in vitro-Test)
GLP compliance:
yes
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Target gene:
Chromosome aberration assay.
Species / strain / cell type:
mammalian cell line, other: Chin. Hamster, V79-Zellen
Metabolic activation:
with and without
Metabolic activation system:
S9 Fraktion isoliert aus Lebern von Aroclor 1254 induzierten männlichen Sprague Dawley Ratten "ENGLISH" S9 fraction isolated from livers of Aroclor 1254 induced male Sprague Dawley rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation, 18 h recovery): 0, 12, 15, 18 µg/ml
Concentration range in the main test (without metabolic activation 18 h recovery): 0, 64, 96, 128 µg/ml
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: mitomycin C, cyclophosphamide
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours .
Exposure period (without metabolic activation): 4 hours .

Fixation time:
Aufarbeitung nach 18 Stunden:

(alle Konzentrationen +/- S9-Mix)

Aufarbeitung nach 30 Stunden:

(128 µg/ml (- S9 Mix); 18 µg/ml (+ S9 Mix)

"ENGLISH"

Sacrifice after 18 h:

(all concentrations +/- S9 mix)


Sacrifice after 30 h:

(128 µg/ml (- S9 mix); 18 µg/ml (+ S9 mix)
Evaluation criteria:
The classes of structural chromosome damage were defined and recorded using essentially the terminology of Rieger and Michaelis (1967, VEB Gustav Fischer Verlag, Jena)
Statistics:
One-sided chi2-test, Fisher's exact test
Species / strain:
other: Chin. Hamster, V79-Zellen
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:
other: Chin. Hamster, V79-Zellen
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
with S9, 4 h treatment, 18 h harvest time at 18 µg/ml significant increased
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: Signs of precipitation at 128 µg/ml.


"ENGLISH"

precipitations at 128 µg/ml

Without S9 mix no cytotoxic effects were observed, with S9 mix cytotoxic effects were observed at 12 µg/ml and above. Precipitation in the medium occurred at 128 µg/ml and above. For reading without S9 mix: 64, 96, 128 µg/ml and with S9 mix: 12, 15, 18 µg/ml were chosen. The chosen cultures were harvested 18 h after the beginning of the treatment. Additional cultures treated with 128 µg/ml (without S9 mix) and 18 µg/ml (with S9 mix) were harvested 30 h after the beginning of the treatment. In the absence of S9 mix cultures treated with Vulcuren VP KA 9188 showed no biologically relevant and statistically significant increased numbers of aberrant metaphases. However in the presence of S9 mix cultures treated with 18 µg/ml Vulcuren VP KA 9188 showed weakly but biologically relevant and statistically significant increased numbers of aberrant metaphases.

Conclusions:
Interpretation of results:
positive with metabolic activation.
negative without metabolic activation.
Executive summary:

In a mammalian test system with Chinese hamster V79 cells toxic and clastogenic effects of Vulcuren VP KA 9188 were seen. The test substance was evaluated in the chromosome aberration assay with or without metabolic activation. In presence of metabolic activation (S9 mix) cytotoxicity was indicated at 12 µg/ml and higher. In addition, cultures of the highest dose group (18 µg/ml, with S9-mix, 18 h harvest time) showed a weak but biological relevant and statistical significant increase in aberrant metaphases. In the absence of metabolic activation no cytotoxic and genotoxic effects were seen up to the highest concentration (128 µg/ml) evaluated (Bayer 2000b).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Two in-vivo tests ( Mammalian erythrocyte micronucleus test and a Comet assay) are available.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study, study design according to an internationally accepted protocol for the comet assay in vivo
Principles of method if other than guideline:
Study was performed according to an internationally accepted protocol for the comet assay in vivo (Hartmann et al., Mutagenesis vol. 18, no. 1, 45-51, 2003).
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: 218-272 g
- Housing: animals were kept individually in type IIA cages
- Water:ad libitum
- Acclimation period: at least 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5°C
- Humidity (%): 40% to 70%
- Air changes (per hr): 10 times per hour
- Photoperiod: 12 hrs dark / 12 hrs light


Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used:
Details on exposure:
The test substance was administered in a volume of 20 ml/kg.
Duration of treatment / exposure:
Number of application: 1, rats were sacrificed 3 h after administration.
Frequency of treatment:
Once
Post exposure period:
3 h
Remarks:
Doses / Concentrations:
0, 2000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
400 mg/kg EMS (administration volume: 10 ml/kg)
Tissues and cell types examined:
Liver and stomach
Details of tissue and slide preparation:
Liver perfusion in situ, preparation of hepatocytes
preparation of stomach cells
Evaluation criteria:
An assay is considered acceptable if the following criteria are met:
1. Tail length data obtained for a given treatment are acceptable as part of the evaluation if obtained from at least two slides and 100 cells per animal.
2. The positive control EMS should induce a mean tail length increase of at least 30 % compared to the mean tail length value of the vehicle control group.
A test was considered positive if there was a biologically relevant and statistically significant increase of the evaluated parameter(s) in tissue cells of treated animals in comparision to the negative control.
Statistics:
One-sided Wilcoxon rank test
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results dose rage finding study:
on day 2 post administration 1 male showed piloerection. There were no gross pathological findings at necropsy. Histopathologically neither necrotic nor apoptotic cells were seen in stomach and liver cells; based on these results the limit dose of 2000 mg/kg was chosen for the comet assay in male rats.

Clinical Observations: no clinical findings after treatment with the test substance in any rat of the 2000 mg/kg treatment group Tissue Tissue Cytotoxicity:

   Liver    Stomach  
 Dose Group  Absolute Viability* [%]   Relative Viability# [%]   Absolute Viability* [%]  Relative Viability# [%] 
 Vehicle Control  75.4 100  80.5  100 
Vulcuren VP KA 9188200 mg/kg  77.7 101.7  77.5  96.3 
 Positive Control EMS400 mg/kg 75.2  98.4  68.4  85.0 

*= mean viability of cell preparation per dose group after perfusion #= relative to vehicle control animals The cell viability of vehicle controls was well within the range of defined cell quality criteria. The viability of liver, and stomach cells of rats exposed to Vulcuren VP KA 9188 was comparable to the respective control value. The same applied to the cells of the evaluated tissues of the positive control animals. Comet assay: Mean tail length per dose group

 Dose Group  Liver Mean Tail Length [µm] +/- SD Stomach  Mean Tail Length [µm] +/- SD
 Vehicle Control  22.23 +/- 1.7  21.12 +/- 0.8
 Vulcuren VP KA 91882000 mg/kg  24.12 +/- 2.2  23.07** +/- 1.5
 Positive ControlEMS 400 mg/kg  34.44** +/-2.3  33.48** +/- 3.0

** p<0.01 (one-sided Mann-Whitney-Wilcoxon)

After administration of 2000 mg/kg Vulcuren VP KA 9188 to male rats the tail lengths of liver cells was comparable to values of the cells of the respective tissue vehicle control animals. The mean comet tail length of stomach cells of the rats treated at 2000 mg/kg Vulcuren VP KA 9188 were statistically significantly different as compared to concurrent control mean value. However, the observed tail length values of stomach cells are comparable to historical control data (see overall remarks). In addition, the individual animal tail length data (see overall remarks) showed that the statistically significant of mean values is not biologically relevant.

In summary, this difference is thus not considered to be biologically relevant.

The tail length of the positive control rats was clearly increased demonstrating the sensitivity of the test system for the detection of genotoxic effects.

Conclusions:
Interpretation of results: negative
Executive summary:

Male Wistar rats, which were treated with 2000 mg/kg Vulcuren VP KA 9188, were evaluated in the Comet assay. The treated rats showed no signs of toxicity. After single application of 2000 mg/kg test substance the tail lengths of liver cells as well the tail lengths of stomach cells were not biologically relevant increased. Based on these findings, the author concluded that Vulcuren VP KA 9188 is non-gentoxic in the comet assay in vivo to liver and stomach cells of male Wistar rats (Lanxess GmbH, 2005).

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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: GLP and OECD Guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: 92/69/EWG, B.12 (Mikrokerntest)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, borchen
- Age at study initiation: 6 to 12 weeks
- Weight at study initiation: 36-41 g
- Housing: animals were kept singly in type I cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at leat 5 d


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5°C
- Humidity (%): 40% to 70%
- Air changes (per hr):
- Photoperiod: 12hrs dark / 12 hrs light


Route of administration:
intraperitoneal
Vehicle:
Maiskeimöl

"ENGLISH"

corn oil
Details on exposure:
Vulcuren Versuchsprodukt KA 9188 was suspended in corn oil using a microdismembrator for 5 minutes, and formed yellowish turbid viscous suspensions. The suspensions were stirred with a magnetic mixer during administration and injected intraperitoneally.
The volume administered was 20 ml/kg bw for treatment group and negative control, 10 ml/kg for the postive control
Duration of treatment / exposure:
Twice
Frequency of treatment:
Twice
Post exposure period:
24 h
Remarks:
Doses / Concentrations:
0, 1000, 2000, 4000
Basis:
nominal conc.
No. of animals per sex per dose:
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 4000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (20 mg(kg)
Tissues and cell types examined:
Bone marrow from femur
Details of tissue and slide preparation:
DETAILS OF SLIDE PREPARATION: Schmidt's method was used to produce the smear (1975, Mutat. Res., 31, 9-15)
Evaluation criteria:
An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/ or available literature data.
Statistics:
Wilcoxon's non-parametric rank sum test
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Doses producing toxicity: >= 1000 mg/kg bw.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Observations: No deaths occurred. Toxic effects: apathy, piloerection, cramps.

Clinical observations at 1000 mg/kg and higher:

Apathy, roughened fur, spasm and periodically stretching of body; these symptoms demonstrate relevant systemic exposure of males to Vulcuren Versuchsprodukt KA 9188.

Mortality: no substance induced mortality.

Ratio polychromatic (PCE) to normochromatic erythrocytes (NCE):

The ratio of polychromatic to normochromatic erythrocytes in males was not biologically relevant altered by the treatment with Vulcuren Versuchsprodukt KA 9188 compared to the negative control (control: 2000: 1938, 1000 mg/kg: 2000: 1894, 2000 mg/kg: 2000: 2121, 4000 mg/kg: 2000: 2456).

No biologically relevant and statistically significant increase of micronucleated polychromatic erythrocytes was noted in the Vulcuren Versuchsprodukt KA 9188 treated animals compared to the negative control

Incidence of micronucleated PCE:

negative control: 3.0/2000

2 x 1000 mg/kg bw: 2.2

2 x 2000 mg/kg bw: 3.0

2 x 4000 mg/kg bw: 2.4

In addition, no biologically relevant variation between the negative control and the treatment groups were noted in the number of micronucleated normochromatic erythrocytes.

The positive control, CPA, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 28.4/2000, which represents biologically relevant increases in comparison to the negative control.

Conclusions:
Interpretation of results: negative
Executive summary:

The test substance Vulcuren VP KA 9188 was evaluated in the in vivo micronucleus assay with male NMRI mice. The test substance was applied at concentrations of 0, 1000, 2000, 4000 mg/kg body weight. All animals from the treatment groups showed clinical signs like apathy, roughened fur, spasm and periodically stretching of the body, which demonstrated a relevant systemic exposure of males to Vulcuren VP KA 9188. The ratio of polychromatic to normochromatic erythrocytes in males was not biologically relevant altered by the treatment with the test substance compared to the negative control. In addition, no biologically relevant and statistically significant increase of micronucleated polychromatic erythrocytes was noted in the treated animals compared to the negative control (Bayer AG 2000c).

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

Additional information

Non-human information

In vitro data

The test substance Vulcuren (purity: 99.7%) was evaluated in a Salmonella/microsome assay according to current guidelines. Vulcuren (purity: 99.7%) was considered to be non-mutagenic with and without metabolic activation in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test. No toxic effects were seen in any of the dose groups evaluated (Bayer 2000a). These findings were confirmed by a previous bacterial study, which also detected a non-mutagenic and non-toxic potential of the test substance (Hüls AG 1992d).

However, in a mammalian test system with Chinese hamster V79 cells toxic and clastogenic effects of Vulcuren (purity: 99.7%) were seen. The test substance was evaluated in the chromosome aberration assay with or without metabolic activation. In presence of metabolic activation (S9 mix) cytotoxicity was indicated at 12 µg/ml and higher. In addition, cultures of the highest dose group (18 µg/ml, with S9-mix) showed a weak but biological relevant and statistical significant increase in aberrant metaphases. In the absence of metabolic activation no cytotoxic and genotoxic effects were seen up to the highest concentration (128 µg/ml) evaluated (Bayer 2000b).

No data from in vitro mammalian mutation assays are available. However, data from appropriate in vivo mammalian tests are available (see below).

In vivo data

The test substance Vulcuren (purity: 99.7%) was evaluated in the in vivo micronucleus assay with male NMRI mice. The test substance was applied at concentrations of 0, 1000, 2000, 4000 mg/kg body weight. All animals from the treatment groups showed clinical signs like apathy, roughened fur, spasm and periodically stretching of the body, which demonstrated a relevant systemic exposure of males to Vulcuren (purity: 99.7%). The ratio of polychromatic to normochromatic erythrocytes in males was not biologically relevant altered by the treatment with the test substance compared to the negative control. In addition, no biologically relevant and statistically significant increase of micronucleated polychromatic erythrocytes was noted in the treated animals compared to the negative control (Bayer AG 2000e). Thus, no clastogenic effects of the test substance could be detected in the in vivo micronucleus assay.

The non-genotoxic potential of Vulcuren was confirmed in an in vivo Comet assay. Male Wistar rats were treated once via gavage with 2000 mg/kg Vulcuren (purity: 96.2%). The treated rats showed no signs of toxicity. After a single application of 2000 mg/kg test substance the tail lengths of liver cells as well the tail lengths of stomach cells were not biologically relevant increased. Based on these findings, the author concluded that Vulcuren (purity: 96.2%) is non-genotoxic in the comet assay in vivo to liver and stomach cells of male Wistar rats (Lanxess GmbH 2005).


Endpoint Conclusion: No adverse effect observed (negative).

Justification for classification or non-classification

The test substance Vulcuren was non-mutagenic in bacteria, whereas in a mammalian cell test system a weakly positive effect was detected. The increase of aberrant metaphases seen in Chinese hamster V79 cells in presence of metabolic activation was considered as biologically relevant and statistically significant. However, these clastogenic effects could not be confirmed in two additional in vivo genotoxicity assays. No clastogenic effects were detected in an in vivo micronucleus assay, beside the fact that concentrations up to 4000 mg/kg bw were evaluated and systemic toxicity was indicated. In addition, an in vivo indicator assay, the comet assay, which detects DNA damages like DNA single- and DNA double-strand breaks, which are precursor of clastogenic lesions, was negative. In conclusion, according to the results from the in vivo assay the test substance Vulcuren (purity: > 96%) is considered to be non-mutagenic.

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not justified.