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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Reactive Blue FC 15353 has been tested in 2 in vitro assays, namely, reverse gene mutation assay in bacteria (OECD 471) and a gene mutation assay in Chinese Hamster V79 Cells (OECD 476) plus 1 in vivo assay for cytogenicity, the micronucleus test in mouse (OECD 474). The test item was negative in all three test systems.
Link to relevant study records
Reference
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
Study period:
1985-Sep-16 to 1985-Oct-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and Guideline Study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
No major deviations
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8-12 weeks
- Weight at study initiation:25-37 g
- Assigned to test groups randomly: yes, under following basis: according to a plan from the Institute for Biometry, Bayer AG, Wuppertal
- Housing: 3 or 5 animals (males/females separated) per macrolon cage (type I or III) on wood granulate
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: at least 7 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22 °C
- Humidity (%): 60-90 %
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light
Route of administration:
oral: gavage
Vehicle:
test substance: 0.5 % aqueous Cremophor-emulsion
positive control: deionized water
negative control: 0.5 % aqueous Cremophor-emulsion
Details on exposure:
volume of oral application:
test substance and negative control: 20 ml/kg body weight
positive control: 10 ml/kg body weight
Duration of treatment / exposure:
test substance: 24, 48 and 72 hours
positive and negative control: 24 hours
Frequency of treatment:
single dose
Remarks:
Doses / Concentrations:
7500 mg/kg body weight
Basis:
no data
No. of animals per sex per dose:
5/sex
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): well-known cytostatic drug and clastogene with bifunctional alcylating effect
- Route of administration: oral, gavage
- Doses / concentrations: 20 mg/kg body weight
Tissues and cell types examined:
bone marrow of femur
1000 polychromatic erythrocytes counted per animal plus number of mature eryhtrocytes per 1000 polychromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based of a pretest with 5000 mg/kg body weight and 7500 mg/kg body weight (5 animals each); test substance application: gavage; single dose.
Both doses caused clinical symptoms within 72 hours, i.e. somnolescence, blue discoloration of skin and feces, pale eyes and bristled fur. No death occured.

DETAILS OF SLIDE PREPARATION:
erythrocytes were prepared according to the method reported by W. Schmid, Mutation Res. 31, 9-15, 1975
samples were stained using a slide stainer Typ Haematek, Fa. Miles and than wahsed with methanol and water, and subsequently dried
prior to analysis, samples were covered by glass after 10 min incubation in xylene

METHOD OF ANALYSIS:
1000 polychromatic erythrocytes and mature eryhtrocytes per 1000 polycrhomatic erythrocates were counted using a light microscop with 1000x magnification.
Evaluation criteria:
The test substance is considered positive if the mean number of micronucleated polychromatic erythocytes of the test substance group is higher than in the negativ control group; and, if so, the significance level calculated by the Wilcoxon signed-rank test is < 5 %.
Statistics:
Numbers of micronucleated polychromated erythrocytes per 1000 polychromated erythrocytes were counted from 5 animals/sex for each exposure time plus negative and positive control and means were calculated. A Wilcoxon signed-rank test was performed when the mean of the test substance was higer than the negative control.
Additional the 1s-intervals were calculated.
The usage of the Wilcoxon signed-rank test is appropiate; the significance level of < 5 % is appropiate.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
somnolescence, blue discoloration of skin and feces, pale eyes and blistered fur
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
the test item had no effect on erythopesis.
Conclusions:
Interpretation of results (migrated information): negative
Reactive Blue FC 15353 did not cause an increase of micronucleated polychromatic erythrocytes and is therefore considered not-clastogenic in the micronucleus in vivo test.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay, (5/sex) were treated once by gavage with Reactive Blue FC 15353 at a dose of 7500 mg/kg bw. Bone marrow cells were harvested at 24, 48, and 72 hours post-treatment. The vehicle was 0.5 % aqueous cremophor-emulsion.

There were signs of toxicity (somnolescence, blue discoloration of skin and feces, pale eyes and blistered fur) during the study. Reactive Blue FC 15353 was tested at an adequate dose based on pretests with 5000 mg/kg bw and 7500 mg/kg bw. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. It satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

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

Additional information

Additional information from genetic toxicity in vivo:

In a reverse gene mutation assay in bacteria, strains TA 1535, TA 1537, TA 100 and TA98 of S. typhimurium were exposed to Reactive Blue FC 15353, solved in water at concentrations of 20, 100, 500, 2500, 12400 µg/plate (first experimental set) and 775, 1550, 3100, 6200, 12400 µg/plate (second experimental set) in the presence and absence of mammalian metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains.There was no evidence of induced mutant colonies over background, wherefore Reactive Blue FC 15353 was considered not genotoxic in this test.

In an in vitro gene mutation assay Chinese Hamster V79 cells were exposed to a structural analogue of Reactive Blue FC 15353 at concentrations ranging from 250 to 75000 µg/mL. Relevant cytotoxic effects, indicated by a relative cloning efficiency or a relative cell density at first subcultivation of less than 50 %, occurred without metabolic activation at test substance concentrations 2000 µg/mL (experiment I) and 3750 µg/mL (experiment II) and with metabolic activation at concentrations 5500 µg/mL (experiment I and II). The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency or relative cell density was covered with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers per 106 cells was observed in the main experiments up to the maximum concentration. The mutation frequency did not exceed the historical range of solvent controls and the induction factor did not reach or exceed the trreshold of 3.0. Therefore, the test item was considered not mutagenic in this experiment.

Finally, an in vivo Micronucleus test in NMRI mouse using Reactive Blue FC 15353 at 7500 mg/kg body weight was performed. Mice were treated once by gavage and bone marrow cells were harvested 24, 48 and 72 hours post-treatment. Signs of toxicity (somnolescence, blue discoloration of skin and feces, pale eyes and blistered fur) were observed, indicating systemic availability of the test item; no death occurred.The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow after any treatment time. Based on the weight of evidence, the test substance Reactive Blue FC 15353 is not considered genotoxic in an appropriate testing battery.


Justification for selection of genetic toxicity endpoint
In vivo genetic toxicity study according to OECD guideline 474 for in vivo cytogenicity.

Justification for classification or non-classification

Reactive Blue FC 15353 is considered not genotoxic based on the results of two negative in vitro and one negative in vivo test.