Blue MGi 1037-Na (notification presscake)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The structural analogue substance Blue MGi 1037 was found to be clearly non-mutagenic in bacterial tester strains and showed clastogenic effects in mammalian cells in vitro in a moderate to strong cytotoxic range. This clastogenic effect, however, is questionable since the cytogenetic damage is considered a secondary effect linked to the cytotoxicity of the substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 14, 2000 - June 29, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Directive 92/69/EEC, B.14

Version / remarks:

1992

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : liver microsomal fraction
- method of preparation of S9 mix : Livers of 8-12 weeks old male rats, strain Wistar which received three applications of 80 mg/kg bw Phenobarbital i.p. dissolved in aqua deionised and beta-naphthoflavone orally dissolved in corn oil. The livers were prepared 24 h after the last treatment.
S9 liver microsomal fraction was also obtained and used from hamsters. The livers of 7-8 weeks old male Syrian golden hamsters.
- concentration or volume of S9 mix and S9 in the final culture medium : 15% final concentration (rat S9) or 30% (hamster S9)

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 33 - 5000 µg/plate
Concentration range in the main test (without metabolic activation): 33 - 5000 µg/plate

Vehicle / solvent:

Solvent: Bidistilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix, TA1535, TA100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-o-phenylene-diamine

Remarks:

without S9 mix, TA1537, TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methyl methane sulfonate

Remarks:

without S9 mix, TA 102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix, TA1535, TA100, TA1537, TA98, TA102

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

congo red

Remarks:

with hamster S9 mix only, TA 98

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Test substance adde in agar (plate incorporation, experiment I), pre-incubation (experiment II)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 30 min at 30°C
- Exposure duration/duration of treatment: 48 h at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in number of revertants

Evaluation criteria:

A test item is considered positive if either a dose related increase in the number of revertants or a biologically relevant and reproducible increase for at least one test concentration is induced. A test item producing neither a dose related increase in the number of revertants nor a biologically relevant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. A biologically relevant response is described as follows: A test item is considered mutagenic if in strains TA 98, TA 100, and TA 102 the number of reversions is at least twice as high and in strains TA 1535 and TA 1537 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the above described enhancement factors or not.

Statistics:

No statistical evaluation

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

STUDY RESULTS
- see attachement
Slight toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strains TA 1535 (33 µg/plate) and TA 98 (10 µg/plate) and with metabolic activation in strain 1537 (333 and 1000 µg/plate) in experiment I, and at 5000 µg/plate with metabolic activation in strain TA 1537 in experiment II. The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

HISTORICAL CONTROL DATA
- see attachment

Conclusions:

BLUE MGi 1037 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

A study according OECD TG 471 was performed to investigate the potential of BLUE MGi 1037 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 TA 102.

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:

33; 100; 333; 1000; 2500; and 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strains TA 1535 (33 µg/plate) and TA 98 (10 µg/plate) and with metabolic activation in strain 1537 (333 and 1000 µg/plate) in experiment I, and at 5000µg/plate with metabolic activation in strain TA 1537 in experiment II.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BLUE MGi 1037 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 increase 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, BLUE MGi 1037 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 29, 2000 - November 16, 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Directive 92/69/EEC, B.10

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

mammalian cell line, other: Chinese Hamster V79 cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: V79 cell line supplied by Laboratory for Mutagenicity Testing, LMP, Technical University Darmstadt)

For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: MEM supplemented with 10% fetal calf serum
- Periodically checked for karyotype stability: yes
- Number of passages: passage 5-15
- Cell cycle: 12 h
- Modal number of chromosomes: 22

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: The cells were incubated at 37°C in a humidified atmosphere with 4.5% carbon dioxide.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbital/beta-Naphthoflavone induced rat liver S9 mix was used as the metabolic activation system.
- method of preparation of S9 mix : The S9 liver microsomal fraction was obtained from the livers of 8-12 weeks old male rats, strain Wistar HanIbm which received daily applications of 80 mg/kg bw phenobarbital i.p. dissolved in deionized water and beta-naphthoflavone orally dissolved in corn oil on three subsequent days. The livers were prepared 24 h after the last treatment.
- concentration or volume of S9 mix and S9 in the final culture medium : 5% final S9 concentration

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 62.5 - 1000 µg/mL
Concentration range in the main test (with metabolic activation): 312.5 - 1000 µg/mL
Concentration range in the main test (without metabolic activation): 156.3 - 5000 µg/ml
Concentration range in the main test (without metabolic activation): 25 - 2500 µg/mL
Concentration range in the main test (without metabolic activation): 25 - 300 µg/mL

Vehicle / solvent:

Deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 4,18 and 28 hours
In experiment I, the exposure period was 4 h with and without metabolic activation. In experiment II the exposure period was 4 h with S9 mix and 18 h and 28 h without S9 mix. A third experiment was performed to verify the results of experiment II using an exposure period of 18 h in the absence of S9 mix. The chromosomes were prepared 18 h (exp. I, II and III) and 28 h (exp. II and III) after start of treatment with the test item.

FOR CHROMOSOME ABERRATION
- Spindle inhibitor: colchicine, 0.2 µg/mL for 2 hours
- Methods of slide preparation and staining technique used including the stain used: After incubation in the hypotonic solution the cells were fixed with 3+1 methanol + glacial acetic acid. After preparation the cells were stained with Giemsa.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
Evaluation of the cultures was performed using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduced cell numbers

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups are 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 are 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.

Statistics:

Statistical significance was confirmed by means of the Fischer's exact test (p <0.05).

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

4 h treatment / 18 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(4000 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

4 h treatment / 28 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(2500 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

4 h treatment / 18 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(2500 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

18 h treatment / 18 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(>= 200 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

18 h treatment / 28 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(300 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

28 h treatment / 28 h harvest time

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(200 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
Experiment I (with S9) has been repeated due to the lack of toxicity. Experiment IIa (without S9 mix) has been repeated because of the strong toxicity. Experiment IIb (without S9 mix) has been repeated because of a bacterial contamination of the sample.

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: no influence
- Data on osmolality: no influence

RANGE-FINDING/SCREENING STUDIES
In a range finding pre-test on toxicity cell numbers 24 h after start of treatment were scored as indicator for cytotoxicity. Concentrations between 39.1 and 5000 µg/mL were applied in accordance to OECD 473. Clear toxic effects were observed after 4 h treatment with 5000 µg/mL and above in the absence of S9 mix and with 1250 µg/mL and above in the presence of S9 mix. In addition, 24 h continuous treatment with 312.5 µg/mL and above in the absence of S9 mix induced strong toxic effects.

MAIN TEST
- Genotoxicity results: see attachment


HISTORICAL CONTROL DATA
see attachment

Conclusions:

In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro only after continuous exposure in the absence of S9 mix. Using a protocol with pulse treatment and a recovery time, both in the absence and presence of S9 mix, no increased aberration frequency was observed. Therefore, the clastogenic potential of BLUE MGi 1037 must be considered to be inconclusive in this chromosome aberration test.

Executive summary:

The test item BLUE MGi 1037, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. 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 (5000 µg/mL) was chosen with regard to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-experiment. In the absence and presence of S9 mix, clear toxic effects indicated by strongly reduced cell numbers or mitotic indices below 50 % of control were observed. In the absence of S9 mix the mitotic indices were reduced only after continuous treatment whereas reduced cell numbers were observed after 4 and 18 h pulse treatment. Reproducibly increased aberration frequencies were observed only after continuous exposure in the absence of S9 mix. Using exposure protocols with pulse treatment and a recovery period, no relevant increase was observed when tested up to cytotoxic concentrations. Therefore, the biological relevance of the increases observed after continues treatment must be regarded as questionable. No 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 increased aberration frequencies (p < 0.05).

 In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro only after continuous exposure in the absence of S9 mix. Using a protocol with pulse treatment and a recovery time, both in the absence and presence of S9 mix, no increased aberration frequency was observed. Therefore, the clastogenic potential of BLUE MGi 1037 must be considered to be inconclusive in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

There is no evidence for a mutagenic potential in the in vivo micronucleus assay in the mouse.

Applying the weight of evidence from in vitro and in vivo tests, the substance is considered to be non-genotoxic.

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

Study period:

May 28, 2001- July 25, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Directive 200/32/EEC, Annex 4C

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test .

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division, CH-4414 Füllinsdorf
- Age at study initiation: 8-10 weeks
- Weight at study initiation: males mean value 35.1 g and females mean value 26.2 g
- Assigned to test groups randomly: yes
- Housing: single, Makrolon Type II, with wire mesh top
- Diet: ad libitum, pelleted standard diet (Altromin 1324)
- Water: ad libitum, tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21± 4
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: deionized water
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food):
- Storage temperature of food:

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Dose / conc.:

500 mg/kg bw (total dose)

Remarks:

24 h preparation interval

Dose / conc.:

1 000 mg/kg bw (total dose)

Remarks:

24 h preparation interval

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

24 h preparation interval

Dose / conc.:

2 000 mg/kg bw (total dose)

Remarks:

48 h preparation interval

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally
- Doses / concentrations: 40 mg/kg bw

Tissues and cell types examined:

bone marrow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.

TREATMENT AND SAMPLING TIMES
Approximately 18 hours before treatment the animals received no food but water ad libitum. At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle or the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h, and 24 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
Preparation of the Animals:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). At least one slide was made from each bone marrow sample.
Analysis of Cells:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test ) will be used as an aid in evaluating the results However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

nonparametric Mann-Withney test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: 2000 mg/kg/bw, tested on 24 animals. Total reduction of spontaneous activity was registered after 24 hours.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: reduction of spontaneous activity up to 6h after treatment, urine of treated animals was bright green within the first hours post treatment

RESULTS OF DEFINITIVE STUDY
- Ratio of PCE/NCE (for Micronucleus assay): see Attachment
- Statistical evaluation: see Attachment

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, BLUE MGi 1037 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

This study was performed to investigate the potential of BLUE MGi 1037 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item were investigated:

24 h preparation interval: 500,1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that BLUE MGi 1037 had no cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, BLUE MGi 1037 is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames Test

A study according OECD TG 471 was performed to investigate the potential of the structural analogue BLUE MGi 1037 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 TA 102.

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:

33; 100; 333; 1000; 2500; and 5000 µg/plate.

Slight toxic effects, evident as a reduction in the number of revertants, were observed without metabolic activation in strains TA 1535 (33 µg/plate) and TA 98 (10 µg/plate) and with metabolic activation in strain 1537 (333 and 1000 µg/plate) in experiment I, and at 5000µg/plate with metabolic activation in strain TA 1537 in experiment II.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with BLUE MGi 1037 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 increase 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, BLUE MGi 1037 is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Chromosome aberration Test

The structural analogue BLUE MGi 1037, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in three independent experiments. 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 (5000 µg/mL) was chosen with regard to the current OECD Guideline 473.

Dose selection of the cytogenetic experiments was performed considering the toxicity data of the pre-experiment.

In the absence and presence of S9 mix, clear toxic effects indicated by strongly reduced cell numbers or mitotic indices below 50 % of control were observed. In the absence of S9 mix the mitotic indices were reduced only after continuous treatment whereas reduced cell numbers were observed after 4 and 18 h pulse treatment.

Reproducibly increased aberration frequencies were observed only after continuous exposure in the absence of S9 mix. Using exposure protocols with pulse treatment and a recovery period, no relevant increase was observed when tested up to cytotoxic concentrations. Therefore, the biological relevance of the increases observed after continues treatment must be regarded as questionable. No 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 increased aberration frequencies (p < 0.05).

 In conclusion, it can be stated that in the study described and under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro only after continuous exposure in the absence of S9 mix. Using a protocol with pulse treatment and a recovery time, both in the absence and presence of S9 mix, no increased aberration frequency was observed. Therefore, the clastogenic potential of BLUE MGi 1037 must be considered to be inconclusive in this chromosome aberration test.

Micronucleus Test

A study was performed to investigate the potential of thestructural analogue BLUE MGi 1037 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised water. Deionised water was used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The following dose levels of the test item were investigated:

24 h preparation interval: 500,1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of NCEs was not substantially increased as compared to the mean value of NCEs of the vehicle control thus indicating that BLUE MGi 1037 had no cytotoxic effectiveness in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, BLUE MGi 1037 is considered to be non-mutagenic in this micronucleus assay.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.