[No public or meaningful name is available]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

FAT 40851 is not mutagenic in ames test but showed positive results in chromosome aberration assay.

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:

Experiment start date - 30 November 2010; Experiment completion date - 23 December 2010; Study completion date - 21 February 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Kanpoan No. 287 -- Environment Protection Agency" "Eisei No. 127 -- Ministry of Health &Welfarew "Heisei 09110131 Kikyoku No. 2 -- Ministry of International Trade & Industry"

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

The bacterial strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Regular checking of the properties of the strains regarding the membrane permeability and ampicillin resistance as well as spontaneous mutation rates is performed in Harlan CCR according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.

Storage The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 %DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S9 (Preparation by Harlan C C R) Phenobarbital/ -Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany), weight approx. 220 - 320 g induced by applications of 80 mg/kg b.w. Phenobarbital i.p. (Desitin; D-22335 Hamburg) and β -Naphthoflavone p.o. (Aldrich, D-89555 Steinheim) each on three consecutive days. The livers are prepared 24 hours after the last treatment. The S9 fractions are produced by dilution of the liver homogenate with a KCl solution (1+3) followed by centrifugation at 9000 g. Aliquots of the supernatant are frozen and stored in ampoules at -80 °C. Small numbers of the ampoules can be kept at -20 °C for up to one week. Each batch of S9 mix is routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration in the S9 preparation was 34.1 mg/mL (lot no. R 100909) in both experiments.

S9 Mix: Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 co-factor solution. The amount of S9 supernatant was 10 % v/v in the S9 mix. Cofactors are added to the S9 mix to reach the following concentrations in the S9 mix: 8 mM MgCl2; 33 mM KCl ; 5 mM Glucose-6-phosphate; 4 mM NADP in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath. The S9 mix preparation was performed according to Ames et al.

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

Deionised water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation - TA 1535, TA 100 - 10 µg/plate

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

Without metabolic activation - TA 1537 (50 µg/plate), TA 98 (10 µg/plate.-

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation - E.coli WPA uvrA (3 µl/plate)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation - 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100), 10 µg/plate (WP2 uvrA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Triplicate
- Number of independent experiments: Two

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approx. 72 h

NUMBER OF REPLICATIONS: 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of revertants (below the indication factor of 0.5)

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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. They showed a distinct increase of induced revertant colonies.

Table 1: Summary of results – pre-test and experiment 1

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
without	water		18±	11±1	31±3	136±21	57±5
	untreated		17±4	12±7	26±5	122±14	47±6
	test substance	3	17±2	13±4	30±4	125±6	59±2
		10	18±7	12±3	32±4	123±12	52±7
		33	15±3	14±2	32±3	131±4	52±10
		100	14±3	12±2	33±9	141±11	60±8
		333	15±2	11±4	29±3	129±17	47±7
		1000	13±5	9±2	28±0	143±6	57±5
		2500	13±1	11±2	28±4	133±13	50±11
		5000	9±1	8±2	24±4	146±10	44±16
	NaN3	10	2058±37 *			2263±17 *
	4-NOPD	10			264±15 *
	4-NOPD	50		93±20 *
	MMS	3.0					1296±55 *
with	water		19±4	15±1	44±4	135±4	62±3
	untreated		18±8	20±3	44±	136±16	53±2
	test substance	3	19±4	17±2	49±40	126±5	49±9
		10	17±2	16±2	37±4	143±19	61±9
		33	19±3	13±3	36±4	152±21	63±7
		100	15±4	13±4	41±9	149±18	70±10
		333	14±1	21±1	36±9	135±12	60±9
		1000	13±2	15±2	39±4	149±15	63±8
		2500	14±2	9±4	33±5	138±17	66±4
		5000	18±6	11±4	31±4	136±11	59±3
	2-AA	2.5	462±10 *	347±62 *	2268±122 *	2101±19 *
	2-AA	10					298±13 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control

 

Table 2: Summary of results –experiment 2

Metabolic activation	Test group	Dose level [µg/plate]	Revertant colony counts (mean ± SD)
			TA1535	TA1537	TA98	TA100	WP2uvrA
without	water		17±4	11±3	35±5	133±12	56±5
	untreated		14±1	14±4	28±3	139±6	56±8
	test substance	33	17±3	12±1	25±4	132±3	50±11
		100	18±6	11±5	31±4	125±11	45±7
		333	19±2	14±1	33±6	130±7	52±10
		1000	16±1	10±3	28±4	117±3	54±11
		2500	15±2	10±2	29±4	131±1	48±
		5000	13±3	9±1	28±7	118±9	404
	NaN3	10	1759±98 *			1825±106 *
	4-NOPD	10			309±12 *
	4-NOPD	50		127±6 *
	MMS	3.0					354±93 *
with	water		15±2	18±3	39±9	140±16	65±3
	untreated		17±4	17±2	42±7	137±11	70±6
	test substance	33	18±7	15±1	42±11	135±14	69±2
		100	18±3	18±5	41±14	122±21	65±1
		333	14±2	17±3	32±5	142±14	59±3
		1000	15±	17±3	28±6	129±11	60±5
		2500	14±2	14±3	27±3	133±17	59±5
		5000	10±3	9±3	26±6	135±8	58±2
	2-AA	2.5	349±2 *	258±23 *	1742±149 *	2668±419 *
	2-AA	10					411±39 *

NaN3: sodium azide, 4-NOPD: 4-nitro-o-phenylene-diamine, MMS: methyl methane sulfonate

2-AA: 2-aminoanthracene

* Value exceeds the respective threshold of twice (TA98, TA100, WP2uvrA) or thrice (TA1535, TA1537) the respective solvent control.

Conclusions:

In conclusion, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

This study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14 under GLP. 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:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

 

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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, test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli 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:

Experiment start date - 03 February 2010; Experiment completion date - 05 August 2010; Study completion date - 29 September 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

"Kanpoan No. 287 -- Environmental Agency" "Eisei No. 127 -- Ministry of Health & Welfare" "Heisei 0911 0131 Kikyoku No. 2 - Ministry of International Trade & Industry".

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts, Hepes (25 mM) and 10 % (v/v) fetal bovine serum (FBS) (medium supplemented with Neomycin (5 µg/mL) and Amphotericin B (2.5 µg/mL).

- Properly maintained: yes:
- stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP); Technical University Darmstadt, 64287 Darmstadt, Germany) stored in liquid nitrogen in the cell bank of Harlan CCR, allowing the repeated use of the same cell culture batch in experiments.
- Periodically checked for Mycoplasma contamination: yes (before freezing)
- Periodically checked for karyotype stability: yes(before freezing)
- Periodically "cleansed" against high spontaneous background: yes
- Propagation:
- stock cultures propagated at 37 °C in 80 cm² plastic flasks
- about 5 x 10e5 cells per flask seeded in 15 mL of supplemented MEM (se above under identity of media).
- cells sub-cultured twice a week
- cell cultures incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (95.5 % air).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9:
S9 (Preparation by Harlan CCR) Phenobarbital/ -naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar rats (Hsd Cpb: WU, Harlan Laboratories GmbH, 33178 Borchen, Germany) weight approx. 220 - 320 g induced by intraperitoneal applications of 80 mg/kg b.w. phenobarbital (Desitin, 22335 Hamburg, Germany) and by peroral administrations of 80 mg/kg b.w. β -naphthoflavone (Sigma-Aldrich Chemie GmbH, 82024 Taufkirchen, Germany) each, on three consecutive days. The livers were prepared 24 hours after the last treatment. The S9 fractions were produced by dilution of the liver homogenate with a KCl solution (1+3 parts) followed by centrifugation at 9000 g. Aliquots of the supernatant were frozen and stored in ampoules at –80 °C. Small numbers of the ampoules were kept at –20 °C for up to one week. Each batch of S9 mix was routinely tested with 2-aminoanthracene as well as benzo(a)pyrene. The protein concentration was 35.0 mg/mL (Lot no. 070110) for Experiment I and IIA, 34.4 mg/mL (Lot no. 260210) for Experiment IIB and 33.7 mg/mL (Lot no. 210510) for Experiment III.

S9 Mix: The S9 mix preparation was performed according to Ames et al. Briefly, an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2(8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4). During the experiment the S9 mix was stored on ice.

Test concentrations with justification for top dose:

Experiment I without S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL
Experiment I with S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL
Experiment II A without S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL
Experiment II A with S9 mix: 39.1, 78.1, 156.3, 312.5, 625.0, 937.5, 1250.0, 1875.0, 2500.0 and 5000.0 μg/mL
Experiment II B without S9 mix: 156.3, 312.5, 625.0, 1250.0, 2500.0, 3000.0, 4000.0 and 5000.0 μg/mL
Experiment II B with S9 mix: 78.1, 156.3, 312.5, 625.0, 1250.0, 1875.0, 2500.0 and 5000.0 μg/mL
Experiment III with S9 mix: 80.0, 120.0, 160.0, 200.0, 240.0, 280.0, 320.0, 360.0, 400.0 μg/mL

- dose selection of Experiment II influenced by test item toxicity determined in Experiment I
- cytotoxic effects observed in the presence of S9 mix following treatment with 312.5 and 1250.0 µg/mL.
- however in even higher concentrations of 2500.0 and 5000.0 µg/mL no cytotoxic effects. Therefore, 5000.0 µg/mL chosen as top treatment concentration in the absence and presence of S9 mix in Experiment II.
- conduction of a confirmatory experiment (Experiment III) on sponsors request to better evaluate the concentration range of beginning cytotoxicity
- cytogenetic evaluation performed following 4 hour treatment in the presence of S9 mix at a 18 hour preparation interval

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Nutrient media

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation - 600 µg/mL (4.8 mM) Experiment IIA and IIB; 1000 µg/mL (8.0 mM) Experiment I

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Saline

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation - 1.0 µg/mL (3.5 µM) Experiment IIB; 1.4 µg/mL (5.0 µM) Experiment I, IIA & III

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
- Experiment I (with and without S9 mix): 4 h
- Experiment IIA (without S9 mix): 18 h (= expression time)
- Experiment IIA (with S9 mix): 4 h
- Experiment IIB (without S9 mix): 18 h (= expression time)
- Experiment IIB (with S9 mix): 4 h
- Experiment III (with S9 mix): 4 h
- Expression time (cells in growth medium):
- Experiment I (with and without S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)
- Experiment IIA (without S9 mix): 18 h (= expression time)
- Experiment IIA (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)
- Experiment IIB (without S9 mix): 18 h (= expression time)
- Experiment IIB (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)
- Experiment III (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate

NUMBER OF CELLS EVALUATED: at least 100 metaphases per culture (exception: positive control in experiment 1: only 50)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell numbers (counting 10 defined fields per slide)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test item is classified as non-clastogenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory’s historical control data range 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 is not in the range of the laboratory’s historical control data range (see Table 3).
and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

Statistical significance was confirmed by means of the Fisher’s exact test (8) (p < 0.05).

However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:

A test item can be classified as aneugenic if
- the number of induced numerical aberrations is not in the range of the laboratory’s
historical control data range (see Table 3).

Acceptability of the test
The chromosome aberration test performed in our laboratory is considered acceptable, if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of the laboratory’s historical control data.
b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

Statistics:

See above under Evaluation criteria.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

a statistically significant increase in the number of aberrant cells excluding gaps after 4h incubation with activation in experiments II A & B and III (6.3 % & 5.3 % at 156.3 µg/mL and 6.0 % at 280 µg/mL respectively).

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 312.5 to 1250 µg/mL with activation, but not at higher concentrations (see"Additional informations on results" for details)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 312.5 to 1250 µg/mL with activation, but not at higher concentrations (see"Additional informations on results" for details)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Exp. I: solvent control 283 mOsm, pH 7.3 versus 309 mOsm and pH 7.2 at 5000.0 µg/mL
- Exp. IIA: solvent control 285 mOsm, pH 7.4 versus 314 mOsm and pH 7.3 at 5000.0 µg/mL
- Exp. IIB: solvent control 286 mOsm, pH 7.4 versus 317 mOsm and pH 7.3 at 5000.0 µg/mL
- Evaporation from medium: no
- Water solubility: no precipitation observed up to 5000.0 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
see Tables 1 - 3

ADDITIONAL INFORMATION ON CYTOTOXICITY:
cytotoxicity seen only with activation at the following concentrations:
- Experiment I (with S9 mix, 4 h of exposure time): 312.5 - 1250 µg/mL, but no cytotoxicity at higher doses
- Experiment IIA (with S9 mix, 4 h of exposure time): 312.5 - 1250 µg/mL, but no cytotoxicity at higher doses
- Experiment IIB (with S9 mix, 4 h of exposure time): 312.5 - 1250 µg/mL, but no cytotoxicity at higher doses
- Experiment III (with S9 mix, 4 h of exposure time): 320 and 400 µg/mL, not tested at higher doses

- Table 2: Number of cells in % of solvent control

Without S9 mix
Experiment I: 4 hrs exposure				Experiment IIA: continuous exposure
Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control	Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control
18 hrs	Solvent control	823	100.0	18 hrs	Solvent control	878	100.0
"	19.5	742	90.2	"	19.5	1014	115.5
"	39.1	749	90.9	"	39.1	980	111.6
"	78.1	706	85.7	"	78.1	1051	119.6
"	156.3	927	112.6	"	156.3	810	92.3
"	312.5	1027	124.7	"	312.5	945	107.6
"	625.0	798	96.9	"	625.0	1007	114.7
"	1250.0	880	106.9	"	1250.0	771	87.8
"	2500.0	890	108.1	"	2500.0	674	76.7
"	5000.0	661	80.3	"	5000.0	0	0.0
Experiment IIB: continuous exposure
Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control
18 hrs	Solvent control	444	100.0
"	156.3	522	117.6
"	312.5	515	116.1
"	625.0	469	105.6
"	1250.0	342	77.1
"	2500.0	260	58.5
"	3000.0	162	36.5
"	4000.0	0	0.0
"	5000.0	0	0.0
With S9 mix
Experiment I: 4 hrs exposure				Experiment IIA: 4 hrs exposure
Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control	Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control
18 hrs	Solvent control	843	100.0	28 hrs	Solvent control	888	100.0
"	19.5	896	106.2	"	39.1	966	108.7
"	39.1	872	103.4	"	78.1	968	109.0
"	78.1	882	104.6	"	156.3	839	94.5
"	156.3	687	81.4	"	312.5	485	54.6
"	312.5	486	57.4		625.0	430	48.4
"	625.0	0	0.0	"	937.5	406	45.7
"	1250.0	0	0.0	"	1250.0	384	43.2
"	2500.0	761	90.2	"	1875.0	635	71.5
	5000.0	735	87.1	"	2500.0	716	80.6
				"	5000.0	862	97.1
Experiment IIB: 4 hrs exposure				Experiment III: 4 hrs exposure
Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control	Preparation interval	Concentration in µg/mL	Mean number of cells	Cells in % of solvent control
18 hrs	Solvent control	506	100.0	18 hrs	Solvent control	766	100.0
"	78.1	470	93.0	"	80.0	682	89.1
"	156.3	402	79.5	"	120.0	614	80.1
"	312.5	267	52.7	"	160.0	719	93.9
"	625.0	217	42.8	"	200.0	679	88.7
"	1250.0	258	51.0	"	240.0	468	61.1
"	1875.0	456	90.2	"	280.0	459	59.9
"	2500.0	327	64.7	"	320.0	350	45.7
"	5000.0	385	76.1	"	360.0	452	59.0
				"	400.0	384	50.1

Experimental groups evaluated for cytogenetic damage are shown in bold characters

-Table 3: Percentage of aberrant and polyploid cells in Chinese hamster V79 cell cultures (January 2008 to December 2009)

	Aberrant cells (%)				Polyploid cells (%)
	No. of studies	Range	Mean	Standard deviation	No. of studies	Range	Mean	Standard deviation
Without S9 mix: preparation interval 18 hrs, treatment 4 hrs
Aqueous solv.(1)	21	0.0 - 3.5	1.6	1.0	10	1.2 - 4.2	2.5	0.8
Organic solv.(2)	51	0.0 - 4.0	1.8	1.0	30	1.2 - 4.6	2.7	0.9
Solvent control (total)	72	0.0 - 4.0	1.8	1.0	40	1.2 - 4.6	2.6	0.9
Positive control:400 - 1200 pg/ml	72	8.0 - 54.0	19.1	11.3
Without S9 mix: preparation interval 18 hrs, treatment 18 hrs
Aqueous solv.(1)	17	0.0 - 2.5	1.2	0.7	8	1.6 - 3.3	2.5	0.6
Organic solv.(2)	38	0.0 - 4.0	1.5	0.8	28	0.7 - 3.5	2.6	0.6
Solvent control (total)	55	0.0 - 4.0	1.4	0.8	36	0.7 - 3.5	2.6	0.6
Positive control:400 - 1200 pg/ml	55	6.5 - 57.0	18.6	10.2
With S9 mix: preparation interval 18 hrs, treatment 4 hrs
Aqueous solv.(1)	31	0.0 - 3.5	1.8	0.9	15	2.0 - 4.7	2.9	0.7
Organic solv.(2)	68	0.0 - 4.0	1.7	1.1	41	0.7 - 5.6	2.9	1.1
Solvent control (total)	99	0.0 - 4.0	1.8	1.0	56	0.7 - 5.6	2.9	1.0
Positive control: CPA 1.0 - 2.0 Ng/ml	101	8.0 - 46.0	13.2	5.4

(1)      Aqueous solvents: deionised water (10 % v/v), 0.9 % (w/v) saline (10 % v/v) and culture medium MEM

(2)      Organic solvents: dimethyl sulfoxide, acetone, ethanol and tetrahydrofurane (0.5 % v/v)

Conclusions:

FAT 40851/A induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in the presence of S9 mix.

Executive summary:

The test item FAT 40851/A, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in four independent experiments according to OECD TG 473 and GLP.

Test conditions:

Experiment I without S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL

Experiment I with S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL

Experiment II A without S9 mix: 19.5, 39.1, 78.1, 156.3, 312.5, 625.0, 1250.0, 2500.0 and 5000.0 μg/mL

Experiment II A with S9 mix: 39.1, 78.1, 156.3, 312.5, 625.0, 937.5, 1250.0, 1875.0, 2500.0 and 5000.0 μg/mL

Experiment II B without S9 mix: 156.3, 312.5, 625.0, 1250.0, 2500.0, 3000.0, 4000.0 and 5000.0 μg/mL

Experiment II B with S9 mix: 78.1, 156.3, 312.5, 625.0, 1250.0, 1875.0, 2500.0 and 5000.0 μg/mL

Experiment III with S9 mix: 80.0, 120.0, 160.0, 200.0, 240.0, 280.0, 320.0, 360.0, 400.0 μg/mL

- Exposure duration:

  - Experiment I (with and without S9 mix): 4 h

  - Experiment IIA (without S9 mix): 18 h (= expression time)

  - Experiment IIA (with S9 mix): 4 h

  - Experiment IIB (without S9 mix): 18 h (= expression time)

  - Experiment IIB (with S9 mix): 4 h

  - Experiment III (with S9 mix): 4 h

- Expression time (cells in growth medium):

  - Experiment I (with and without S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)

  - Experiment IIA (without S9 mix): 18 h (= exposure time)

  - Experiment IIA (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)

  - Experiment IIB (without S9 mix): 18 h (= exposure time)

  - Experiment IIB (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)

  - Experiment III (with S9 mix): 14 h (+ 4 h of exposure time = 18 h in total)

 

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation and Experiment IIA with metabolic activation, where only 50 metaphases were evaluated. The highest applied concentration (5000 µg/mL) was chosen with respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data from relevant cytotoxic effects determined as mitotic index or cell numbers of approximately 50 % of control cultures were solely observed after 4 hours treatment in the presence of S9 mix. In the absence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item up to cytotoxic concentrations. In the presence of S9 mix statistically significant and reproducible increases of chromosomal aberrations were observed. Therefore, this effect is judged being biologically relevant. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in Vitro. Therefore, FAT 40851/A is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

FAT 40851 was not clastogenic in the micronucleus study.

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:

Experiment start date - 29 July 2010; Experiment completion date - 25 August 2010; Study completion date - 29 September 2010.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Identification: FAT 40851/A TE
Batch Number: TZ 5891 / BOP 02-09
Purity: 69.9 % all coloured components
Appearance: Orange powder
Expiry Date: July 31, 2014
Storage Conditions: At room temperature at about 20 °C

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system 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 = 36.5 g (SD ± 1.7 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: 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
- 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

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Vehicle control - 24 hr exposure

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose group - 24 hr exposure

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Mid dose group - 24 hr exposure

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose group - - 24 and 48 hr exposure

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):

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 oral 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:

Non-parametric Mann-Whitney test

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

Negative 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 orange 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 orange dye
- testing up to the limit dose

- Table 2: Summary of Micronucleus Test Results

Test group	Dose mg/kg bw	Sampling time (h)	PCEs with micronuclei (%)	Range	PCE per 2000 erythocytes
vehicle	0	24	0.114	1 - 4	1275
test item	500	24	0.064	1 - 2	1240
test item	1000	24	0.100	0 - 5	1211
test item	2000	24	0.086	0 - 6	1166
positive control	40	24	2.736	39 -75	1047
test item	2000	48	0.107	1 - 3	1215

 

- 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 FAT 40851/A/kg b.w.; 24 h	-	n.t.
1000 mg FAT 40851/A/kg b.w.; 24 h	-	n.t.
2000 mg FAT 40851/A/kg b.w.; 24 h	-	n.t.
40 mg CPA/kg b.w.; 24 h	+	0.0003
2000 mg FAT 40851/A/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:

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 item FAT 40851/A 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 in a GLP-certified laboratory. The test item was formulated in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 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 FAT 40851/A TE per kg bw (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 FAT 40851/A 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 FAT 40851/A were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 40 mg/kg bw 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

- In vitro Ames Test

This study was performed to investigate the potential of test substance to induce gene mutations in the plate incorporation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA according to OECD Guideline 471 and EC method B13/14 under GLP. 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:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments. No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test substance 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, test substance is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.

 

InVitro Chromosome abberation study

The test item FAT 40851/A, dissolved in deionised water, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in four independent experiments according to OECD TG 473 and GLP. Dose selection for the cytogenetic experiments was performed considering the toxicity data from relevant cytotoxic effects determined as mitotic index or cell numbers of approximately 50 % of control cultures were solely observed after 4 hours treatment in the presence of S9 mix. In the absence of S9 mix, neither a statistically significant nor a biologically relevant increase in the number of cells carrying structural chromosomal aberrations was observed after treatment with the test item up to cytotoxic concentrations. In the presence of S9 mix statistically significant and reproducible increases of chromosomal aberrations were observed. Therefore, this effect is judged being biologically relevant. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p <0.05) in cells with structural chromosome aberrations. In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, FAT 40851/A TE is considered to be clastogenic in this chromosome aberration test in the presence of metabolic activation.

 

- In vivo micronucleus study

The test item FAT 40851/A TE 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 in a GLP-certified laboratory. The test item was formulated in sterile water, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 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 FAT 40851/A TE per kg bw (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 FAT 40851/A 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 FAT 40851/A TE were near to the value of the vehicle control group. Additionally all values were within the historical vehicle control database. 40 mg/kg bw 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.

 

General:

 

The negative in vivo Micronucleus assay overrules the positive result from the in vitro Chromosome abberation test. The Ames Test is negative and the negative in vivo Micronucleus assay also covers the endpoint of gene mutation in mammalian cells/organisms according to the ECHA helpdesk (see above). Therefore it can be concluded that FAT 40851/A  is neither clastogenic or aneugenic nor mutagenic. Accordingly it can be concluded that FAT 40851/A is not genotoxic.

Justification for classification or non-classification

Based on the above stated assessment of the genotoxic potential FAT 40851/A (Ames test negative, in vitro mammalian chromosome aberration test positive, in vivo mammalian micronucleus test in bone marrow cells negative) the substance is deemed non-genotoxic and accordingly does not need to be classified according to Council Directive 2001/59/EC (28th ATP of Directive 67/548/EEC) and according to CLP (REGULATION (EC) No 1272/2008 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL) as implementation of UN-GHS in the EU.