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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (according to OECD 471, Ames test): negative in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted May 26, 1983; not tested in TA 102 or E.coli WP2 uvrA
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
s9 liver microsomal fraction from rat
Test concentrations with justification for top dose:
Exp. 1 plate incorporation: 3.3 to 100 ug/plate,
Exp. 2: pre-incubation test: 6.6-333 ug/plate (TA 1535, TA1537), 1.0-100 (TA98, TA100)
Vehicle / solvent:
water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water
Positive controls:
yes
Remarks:
positive controls as seen below
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix : TA 1535, TA100
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylene-diamine
Remarks:
TA1537, TA98, without S9mix
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
with S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1: in agar (plate incorporation); Experiment 2: preincubation


DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants


Evaluation criteria:
- negative control in the laboratory historical range for each tester strain
- positive control chemicals produces responses in all tester strains within laboratory historical range
- Test substance is considered as mutagenic if:
- a dose-related and reproducible increase in the number of revertants occurs
- a substantial and reproducible increase for at least one test concentration (TA98, TA100 twofold increase, TA1535, TA1537 threefold increase compared to the corresponding solvent control)
Mean plate count should be at least three times the concurrent vehicle control group mean
- selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate
Statistics:
none
Key result
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:
in concentrations above 100 ug/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Interpretation of results:
negative

Under the conditions tested, the test substance did not cause gene mutations in the genome of the tester strains used.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Genotoxicity in somatic cells in vivo (EU Method B.12, mutagenicity, Micronucleus test): negative for clastogenicity in NMRI mice

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1992
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan-Winkelmann GmbH, Borchen, Germany
- Fasting period before study: 16 hours
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days



Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water (aqua ad niectabilia)
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Duration of treatment / exposure:
single dose
Frequency of treatment:
single dose
Post exposure period:
sampling time 24 and 48 h post application
Remarks:
Doses / Concentrations:
males: 31.6 mg/kg bw., females 21.5 mg/kg bw.
Basis: maximum tolerated dose as determined in a prexperiment
nominal conc.
No. of animals per sex per dose:
negative control: 12
positive control: 6
test material: 14
Evaluation: 7 test material group, 6 negative control, 5 positive control
Control animals:
other: physiological saline solution (0.9 %)
Positive control(s):
- cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 31.6 mg/kg bw
Tissues and cell types examined:
bone marrow smear of the femurs
From each animal 1000 PCEs were scored.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
highest tolerable dose (higher concentrations caused severe toxic symptoms)

METHOD OF ANALYSIS:
scoring under microscope

Evaluation criteria:
test substance is considered mutagenic if it produces a statistically significant (p< 0.05) and reproducible positive response at any of the test points compared to the negative control group
Statistics:
Poisson test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
hypokinesiahypokinesia, tremor, clonic convulsions (females only), decreased muscle tone, loss of righting reflexes. One female animal died.

RESULTS OF RANGE-FINDING STUDY
- Dose range: 31.6 - 38.3 mg/kg bw (males), 21.5-31.6 mg/kg bw (females)
- Solubility: yes
- Clinical signs of toxicity in test animals: severe toxicity symptoms, one female of the high dose died.
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: methodical reasons


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): slightly lower in the test group, no clear myelotoxic effect can be deduced from the findings
control: 2.8/1.6 (24 h), 1.6/3.0 (48 h) ACA: 2.0/1.4 (24 h), 2.0/0 (48 h), values are presented as males/females
Conclusions:
Interpretation of results: negative
No significant test material related increase in micronucleated PCEs was observed. Therefore the substance is considered non mutagenic in the mouse micronucleus assay. In the positive control animals, a significant increase in the number of PCEs was observed and thus validated the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Genetic toxicity in vitro:

In vitro gene mutation in bacteria:

The test item was investigated for the mutagenic potential in a Bacterial Reverse Mutation Assay (Ames test) performed according to OECD guideline 471 and in compliance with GLP (96 -0070 -DGM). S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 were exposed to the test in item in the presence and absence of metabolic activation (S9 mix) in triplicate.

Based on a plate incorporation pre-experiment with and without metabolic activation, concentrations for the main mutation assay were selected at 1.0 – 333 μg/plate. The pre-experiment was reported as experiment I. A pre-incubation test with the same concentrations with and without metabolic activation served as experiment II.Solvent (water) and appropriate positive controls were included. After incubation, plates were inspected for bacterial background lawn and the number of revertant colonies was determined.

The test item did not induce a biologically relevant increase in the number of revertant colonies in any of the tested strains either with or without metabolic up to the highest tested concentration in the presence and absence of S9 mix. Positive and vehicle controls confirmed the validity of the test system and demonstrated the functionality of the S9 mix.

Thus, under the experimental conditions chosen, the test item is not mutagenic in bacteria with and without metabolic activation.

Genetic toxicity in vivo:

In a guideline study performed according to EU Method B.12 on mutagenicity and in compliance with GLP, the test item was dissolved in water and administered orally to NMRI mice (96 -0069-DGM). Based on the results of a preliminary experiment, dose levels for the mutagenicity study were selected. Groups of 14 mice/sex received an application of 21.5 -31.6 and 31.6 -38.8 mg/kg bw for females and males, respectively. A group of 6 animal/sex served as positive control and received 31.6 mg/kg bw cyclophosphamide. 24 and 48 h after the last injection, bone marrow smears were prepared. For each animal, 1000 polychromatic erythrocytes were screened for the presence of micronuclei.

One female animal died prior to the sampling time. Clinical symptoms were hpokinesiahypokinesia, tremor, clonic convulsions  in females only and decreased muscle tone, loss of righting reflexes both sexes.

There were no treatment-related changes in the frequency of micronucleated PCEs. The test item did not induce a statistically significant increase in the frequency of micronucleated PCEs. Ratio of PCE/NCE was slightly lower in the test groups. In the positive control animals, a significant increase in the number of PCEs was observed and thus validated the test. Under the conditions of the mentioned study, the test item has no genotoxic potential in vivo.

Additional information

The available information on genotoxicity in vitro (Ames test) and in vivo (micronucleus test) do not require classification according to Regulation No. (EC) 1272/2008.

No classification for genetic toxicity is warranted according to the criteria of the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations.

Justification for classification or non-classification

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