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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40034 is considered to be not mutagenic based on the negative results in the bacterial reverse mutation assays with FAT 40034 as well as micronucleus assay with FAT 40850.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Justification for type of information:
None
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only 4 tester strains tested; no tester strain to detect crosslinking mutagens was included
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
None
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix from rat liver
Test concentrations with justification for top dose:
0.2, 2, 20, 200, 2000 µg/petri dish
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
Growing and Confirmation of Bacterial Test Strains:
The bacterial strains are kept as frozen broth cultures, in aliquots of 0.5 ml, at -70°C with 8.0* dimethylsulfoxide (DMSO). Fresh cultures are prepared by adding 0.1 ml of a thawed stock culture to 15 ml of nutrient broth (0.8* Difco nutrient broth, 0.5* NaCl). A nutrient agar (2.5% Difco nutrient broth, 1.2* Difco agar) is also streaked with the scrapings. The broth is incubated in the dark in a shaking water bath at 37 deg C for 16 hours, whilst the plate is incubated at 37 C overnight. Broths and plates are then transferred to the refrigerator where they can be kept for one week.

Checking out Tester Strains:
All strains are tested for the presence of their mutations.
a) The mutation in the histidine operon, basic to the test system is tested by checking for growth in the presence of absence of histidine on a minimal medium-agar base.
Bacteria of the nutrient agar plate are streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 mM L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
b) Sensitivity to crystal violet is a check for the presence of the deep rough (rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface. Three drops of the broth culture are spread on the surface of a
nutrient agar plate. A sterile filter paper disc containing crystal violet (10 microlitre of a 1 mg/ml solution) is placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c) Two of the tested strains (98 and 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for its presence, a sterile filter paper disc containing Ampicillin (10 microlitre of 8 mg/ml in 0.02 N NaOH) is placed on a nutrient agar plate spread with the broth. In strains 1535 and 1537 a zone of inhibition occurs around the disc, but for 98 and 100 where the R factor is present, no zone of inhibition is seen.
d) The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation. One drop of the broth is cross-streaked on a nutrient agar plate. One half of the plate is irradiated for 20 seconds under a 15 watt UV lamp at approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.

Induction of rat liver enzymes:
Three male Sprague-Dawley rats weighing approximately 200 grams are given an I.P. injection of Aroclor 125^ (Monsanto) in peanut oil at a
dose of 500 mg/kg.
Five days after the injection, the rats are anaesthetized with ether, decapitated and the livers removed sterilely. The livers are homogenized in 2.0 ml/g tissue sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 x g (Beekman ultracentrifuge). The supernatant (termed the S-9 fraction) is aliquoted into sterile tubes and frozen. These are stored at -70 C.
Rationale for test conditions:
None
Evaluation criteria:
None
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 40034 was not mutagenic for Salmonella typhimurium strains TA 1535, TA1537, TA 98 and TA 100.
Executive summary:

A bacterial reverse mutation assay was performed to determine the mutagenic potential of FAT 40034. Salmonella typhimurium strains TA 1535, TA1537, TA 98 and TA 100 were exposed to the test substance with and without metabolic activation. However, under the conditions employed and using the doubling of the spontaneous reversion rate as a criterion of mutagenicity, FAT 40034/A did not induce significant reverse mutations with and without metabolic activation. Hence, based on the findings of the study, it can be concluded that FAT 40034 was not mutagenic in the bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1979
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
None
Qualifier:
according to
Guideline:
other: AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Deviations:
not specified
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
None
Target gene:
Histidine auxotrophic strains of Salmonella typhimurium
Species / strain / cell type:
other: Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of liver from rats induced with Arochlor and cofactors
Test concentrations with justification for top dose:
None
Vehicle / solvent:
phosphate buffer
Untreated negative controls:
yes
Remarks:
Phosphate buffer
Positive controls:
yes
Positive control substance:
other: With metabolic activation: Strain TA 1535 with cyclophosphamide,
Details on test system and experimental conditions:
The tests were carried out in accordance with the method described by AMES et al. The bacteria on which the tests were performed were the histidineauxotrophic TA 98, TA 100, TA 1535, TA 15 37 and TA 1538, strains of Salmonella typhimurium.
The test was performed with the following concentrations of the trial substance without and with microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 6 75 and 2025 microg/ 0.1 ml. In these experiments tests on Strain TA 15 38 were included. The substance was dissolved in phosphate buffer. Phosphate buffer alone was used for the negative controls. In the experiments in which the substance was metabolically activated, activation mixture was added also. 1 ml activation mixture contains: 0.3 ml S9 fraction of liver from rats induced with Aroclor 1254 and 0.7 ml of a solution of co-factors.

Positive control experiments were carried out simultaneously with the following substances: 1) for Strain TA 98: daunorubicin-HCl (DAUNOBLASTINR), 5 and 10 microg/0.1 ml phosphate buffer; 2) for Strain TA 100: 4-nitroquinoline-N-oxide, 0.125 and 0.25 microg/0.1 ml phosphate buffer; 3) for Strain TA 1535: N-methyl-N1-nitro-N-nitrosoguanidine, 3 and 5 microg/0.1 ml phosphate buffer; 4) for Strain TA 15 37: 9(5) aminoacridine hydrochloride monohydrate, 50 and 100 microg/0.1 ml DMSO; 5) for Strain TA 15 38: 2-nitrofluorene, 5 and 10 microg/0.1 ml DMSO. The activation mixture was tested with Strain TA 15 35 and cyclophosphamide (ENDOXAN-ASTA ), 250 microg/0.1 ml phosphate buffer.
In the experiments with and without the addition of microsomal activation mixture three Petri dishes were prepared per strain and per group (i.e. per concentration or per control group).
The plates were incubated for about 48 hours at 37 deg C in darkness. When the colonies had been counted, the arithmetic mean was calculated.
A test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration .

1.AMESf B.N., F.D. LEE, and W.E. DURSTON (1973), An Improved Bacterial Test System for the Detection and Classification of Mutagens and Carcinogens. Proc. Natl. Acad. Sei. USA 70, 782-786.
2.AMES, B.N., W.E. DURSTON, E. YAMASAKI, and F.D. LEE (1973), Carcinogens are Mutagens: A Simple Test System Combining Liver Homogenates for Activation and Bacteria for Detection.
Proc. Natl. Acad. Sei. USA 7_0, 2281-2285.
3.AMES, B.N., J. McCANN, and E. YAMASAKI (1975), Methods for Detecting Carcinogens and Mutagens with the Salmonella/ Mammalian-Microsome Mutagenicity Test. Mut. Res. 31, 347-364.
Rationale for test conditions:
None
Evaluation criteria:
None
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
In the experiments performed with and without microsomal activation, comparison of the number of histidine-prototrophic mutants in the controls and after treatment with FAT 40034/B revealed no marked differences.
Remarks on result:
other: all strains/cell types tested

None

Conclusions:
FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.
Executive summary:

FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 15 37 and TA 1538. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 675 and 2025 microg/0.1 ml. In these experiments tests on Strain TA 1538 were included.

In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40034/B revealed no marked deviations.

Based on the findings of the study, no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Hence, FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

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

Genetic toxicity in vivo

Description of key information

Read across substance, FAT 40850 did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

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:
2010-07-29 to 2010-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as at 1997
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
as at 2008
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: males mean value = 36.7 g (SD ± 1.5 g)
- Assigned to test groups randomly: not reported
- Fasting period before study: no
- Housing: singly in Makrolon Type II/III cages with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany) and granulated soft wood bedding
(Rettenmaier & Söhne GmbH + Co. KG,73494 Rosenberg, Germany)
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum(Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst, The Netherlands)
- Water (e.g. ad libitum): tap water, ad libitum, (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: >= 5 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 45 - 100
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Source: B. Braun Melsungen AG 34212 Melsungen, Germany
- Catalogue no.: 6724092.00.00
- Justification for choice of solvent/vehicle: chosen for its relative non-toxicity for animals
- Concentration of test material in vehicle: 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in in the vehicle (sterile water).
Duration of treatment / exposure:
single oral gavage
Frequency of treatment:
single oral gavage
Post exposure period:
no
Remarks:
Doses / Concentrations:
main experiment: 500, 1000, 2000 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
toxicity pretest: 2000 mg/kg bw
Basis:

No. of animals per sex per dose:
- main test: 7 males per dose (only males were used in the main study as the toxicity pretest showed comparable susceptibility of males and females)
- toxicity pre-test: 2 males and 2 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
none; no data; cyclophosphamide (CPA)
- Supplier: Fisher Scientific GmbH, 61130 Nidderau, Germany
- Justification for choice of positive control(s): recommended as positive control substance in the OECD guideline
- Route of administration: oral gavage
- Doses / concentrations: 40 mg/Kg bw (concentration in vehicle (water): 4 mg/mL)
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
2000 mg/kg bw is the limit dose according to the OECD guideline. Two lower doses with a spacing factor of 2 were selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
- single aoral treatment
- Sampling:
- pre-test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration
- main test: examination for acute toxic symptoms 1 h, 2-4 h, 6 h, 24 h (and 48 h) after administration; animals of all dose levels sacrificed at 24 h post dosing, an additional high dose group sacrificed 48 post treatment

DETAILS OF SLIDE PREPARATION:
- sacrifices of animals using CO2 followed by bleeding
- removal of the femora, cutting off of epiphyses, marrow flushed out with foetal calf serum using a syringe
- centrifugation of the cell suspension at 1500 rpm (390 x g) for 10 minutes
- discarding of supernatant, resuspesion of the remaining cell pellet and spreading of a small drop of the suspension on a slide
- air drying of the slide and staining with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany)
- mounting of cover slips with EUKITT (Kindler, 79110 Freiburg, Germany)
- preparation of at least one slide per each bone marrow sample

METHOD OF ANALYSIS:
- slide analysis using NIKON microscopes with 100x oil immersion objectives
- analysis for micronuclei of 2000 polychromatic erythrocytes (PCE) per animal
- determination of ratio between polychromatic and normochromatic erythrocytes in the same sample for detection of cytotoxicity
- cytotoxicity expressed in polychromatic erythrocytes per 2000 erythrocytes
- slides were coded
- use of samples from all animals per test group

OTHER:
Evaluation criteria:
The study was considered valid as the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of
micronucleated PCEs compared to the negative control.

Positive result:
If the test item 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.

Negative result
If the test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes it is considered non-mutagenic in this system.
Statistics:
see above under Evaluation criteria
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
The animals treated with 2000 mg/kg b.w. did not express any toxic reactions. However, discoloured urine was observed (orange) in animals treated with 2000 mg/kg bw both in pre-test and main experiment.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Solubility: no problems with solubility reported
- Clinical signs of toxicity in test animals:
- no adverse effects
- orange discoloration of the urine, indicating bioavailability of the test item which is a red dye
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: 2000 mg/kg bw is the limit dose recommended in the OECD guideline.


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable
- Induction of micronuclei (for Micronucleus assay):
- No micronuclei induced in any of the treatment groups
- Micronuclei induced in the positive control group at the expected rate, indicating the sensitivity of the test system
- for details see Tables 2 and 3
- Ratio of PCE/NCE (for Micronucleus assay):
- not significantly changed in any of the treatment groups
- for details see Tables 2 and 3
- Appropriateness of dose levels and route:
- orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a red dye
- testing up to the limit dose
- Statistical evaluation: non-parametric Mann-Whitney test

- Table 2: Summary of Micronucleus Test Results

test group

Dose mg/kg b.w.

Sampling time (h)

PCEs with micronuclei (%)

range

PCE per 2000 erythocytes

vehicle

0

24

0.079

0 - 5

1219

test item

500

24

0.186

0 - 8

1264

test item

1000

24

0.107

1 - 6

1177

test item

2000

24

0.136

2 - 6

1203

positive control

40

24

2.979

23 -82

1118

test item

2000

48

0.057

0 - 2

1233

- Table 3: Statistical significance at the five per cent level (p < 0.05, evaluated by means of the non-parametric Mann-Whitney test).

Vehicle control versus test group

Significance

p

500 mg test material/kg b.w.; 24 h

-

0.0758

1000 mg test material/kg b.w.; 24 h

-

0.2287

2000 mg test material/kg b.w.; 24 h

-

0.0807

40 mg CPA/kg b.w.; 24 h

+

0.0003

2000 mg test material/kg b.w.; 48 h

-

n.t.

-         =         not significant

+         =         significant

n.t. = not tested

- Table 4: Historical controls from 2003 - 2009

 

Negative Controls Males

Positive Controls (CPA) Males

Mean* ± SD

0.096 ± 0.040

2.332 ± 0.696

Range**

0.01 - 0.22

0.70 - 4.52

No. of Experiments

318

316

*:        mean value (percent micronucleated cells)

**:      range of the mean group values (percent micronucleated cells)

Conclusions:
Interpretation of results (migrated information): negative
The test material was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474 (single oral doses of 500, 1000 and 2000 mg/kg bw).
No micronuclei were induced in any of the treatment groups while micronuclei were induced in the positive control group at the expected rate, indicating the sensitivity of the test system. Orange discoloration of the urine in the highest dose group, indicating bioavailability of the test item which is a blue dye.
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 in the bone marrow cells of the mouse.
Executive summary:

The test material was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474.

The test item was formulated in sterile water, which was also 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.

Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. As estimated by a pre-experiment 2000 mg of the test material per kg b.w. (the maximum guideline-recommended dose) was suitable.

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test material did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with 2000 mg/kg b.w. indicating the bioavailability of the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test material were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which

showed a statistically significant 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 in the bone marrow cells of the mouse.

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

Additional information

The genetic toxicity database of FAT 40034 consists of two bacterial reverse mutation assays. However, to complete data requirement at this tonnage level as well as genetic toxicity assessment, a study investigating mutagenic potential in mammalian cells and a study investigating clastogenic potential in mammalian cells is required. The genetic toxicity database of source substance FAT 40850 consists of a bacterial reverse mutation assay, anin vitromammalian cell gene mutation assay, anin vitrochromosomal aberration assay and a micronucleus assay. Hence the studies with source substance were used to complete the genetic toxicity assessment of the target substance.

Mutagenic potential of FAT 40034 was evaluated in two different bacterial reverse mutation assays.

In a key study conducted by Haroz et al. (1978), Salmonella typhimurium strains TA 1535, TA1537, TA 98 and TA 100 were exposed to the test substance with and without metabolic activation. Under the conditions employed and using the doubling of the spontaneous reversion rate as a criterion of mutagenicity, FAT 40034/A did not induce significant reverse mutations with and without metabolic activation. Hence, based on the findings of the study, it can be concluded that FAT 40034 was not mutagenic in the bacterial reverse mutation assay.

In a supporting study conducted by Arni, P. (1979), FAT 40034/B was tested for mutagenic effects on histidine-auxotrophic mutants of Salmonella typhimurium which included TA 98, TA 100, TA 1535, TA 15 37 and TA 1538. The investigations were performed with the following concentrations of the trial substance with and without microsomal activation: 15, 45, 135, 405 and 1215 microg/0.1 ml. A repetition of the experiments was performed with the concentrations of 25, 75, 225, 675 and 2025 microg/0.1 ml. In these experiments tests on Strain TA 1538 were included. In the experiments performed with and without microsomal activation, comparison of the number of back-mutant colonies in the controls and the cultures treated with the various concentrations of FAT 40034/B revealed no marked deviations. Based on the findings of the study, no evidence of the induction of point mutations by FAT 40034/B or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Hence, FAT 40034/B was determined to be non-mutagenic in the bacterial reverse mutation assay.

Data from Read across substance FAT 40850:

Bacterial reverse mutation assay with FAT 40850 (read across)

FAT 40850 was evaluated to induce gene mutations in the plate incorporation test using theSalmonella typhimuriumstrains TA 1535, TA 1537, TA 98, and TA 100, and theEscherichia colistrain WP2 uvrA according to OECD Guideline 471 and EC method B13/14. The assay was performed with and without liver microsomal activation at the concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate. A biologically relevant increase in revertant colony numbers was observed following treatment with the test substance in strains TA 98 and TA 100. Therefore, FAT 40850 is considered to be mutagenic in bacterial cells.

In vitromammalian cell gene mutation assay with FAT 40850 (read across)

In a GLP-compliant mammalian cell gene mutation assay, conducted according to OECD Guideline 476, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation and the potential to induce mutations at the HPRT locus was assessed. No relevant and reproducible increase in mutant colony numbers/106cells was observed in the main experiments at any of the concentrations. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment II at 400 μg/mL without metabolic activation. However, this increase was not reproduced in the parallel culture under identical conditions or in the first experiment without metabolic activation. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. A linear regression analysis was performed to assess a possible dose dependent increase of mutant frequency. A single significant dose dependent trend of the mutation frequency was determined in the second experiment at culture I with metabolic activation. However, the trend was judged as biologically irrelevant since the mutation frequency did not exceed the threshold described above and all of the individual values remained within the historical range of solvent controls. In conclusion, FAT 40850 is considered to be non-mutagenic in this mammalian cell gene mutation assay.

In vitro chromosomal aberration assay with FAT 40850 (read across)

In a GLP-compliant chromosome aberration test, conducted according to OECD Guideline 473, Chinese hamster lung fibroblasts (V79) were exposed to FAT 40850 with and without metabolic activation. In Experiment I and II in the presence of S9 mix, no biologically relevant clastogenicity was observed at the concentrations evaluated either with or without metabolic activation. In Experiment II in the absence of S9 mix statistically significant increases in the number of aberrant cells, excluding gaps were observed at all evaluated concentrations in the range of 180 to 300 µg/mL. All values exceeded the laboratory´s historical solvent control data range (0.0 – 3.5 % aberrant cells, excluding gaps). Therefore, the occurrence of chromosomal aberrations is considered biologically relevant. No increase in polyploid metaphases was noticed after treatment with the test substance as compared to the controls. Hence, it was concluded that the test substance induced structural chromosomal aberrations in V79 cells (Chinese hamster cell line) in vitro.

Micronucleus assay with FAT 40850 (read across)

FAT 40850 was assessed in the micronucleus assay for the potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD TG 474. Seven males per test group were evaluated for the occurrence of micronuclei. The following dose levels of the test substance were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw; 48 h preparation interval: 2000 mg/kg bw. The mean number of polychromatic erythrocytes was not decreased after treatment with the test substance as compared to the mean value of PCEs of the vehicle control indicating that the test substance did not have any cytotoxic properties in the bone marrow. However, the animals showed discoloured urine after treatment with 2000 mg/kg b.w. indicating the bioavailability of the test substance. In comparison to the corresponding vehicle controls, there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test substance. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. In conclusion, it can be stated that the test substance did not induce micronuclei in the bone marrow cells of the mouse.

As discussed above, FAT 40034 was found to be not mutagenic in theSalmonella reverse mutation assays. The source substance, FAT 40850, was found to have mutagenic effect in the bacterial reverse mutation assay, however it could not induce the mutagenicity in the mammalian cell gene mutation assay in vitro. FAT 40850 was found to be clastogenic in a chromosome aberration assay in vitro, but failed to induce micronuclei in the micronucleus assay, hence considered to be not clastogenic. Therefore, based on the negative results in the bacterial reverse mutation assays with the target substance, in vitro mammalian cell gene mutation assay with the source substance as well as micronucleus assay with the source substance, both FAT 40034 and FAT 40850 are considered to be devoid of mutagenic as well as clastogenic potential, and hence to be not genotoxic.

Justification for classification or non-classification

Based on the available data, the substance FAT 40034 does not warrant classification for genotoxicity under the criteria of CLP (1272/2008) Regulation.