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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
No genotoxicity of the test substance found.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 10, 2007 to April 11, 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Commission directive 2000/32/EC, Annex AC, dated May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain: NMRI
Source Harlan Winkelmann GmbH D-33178 Borchen
Number of Animals: 72 (36 males/36 females)
Initial Age at Start of Acclimatisation: 7 - 8 weeks
Acclimatisation: minimum 5 days
Initial Body Weight at Start of Treatment: males mean value 35.6 g (SD ±1.5 g) females mean value 27.6 g (SD ±1.1 g)

Husbandry
The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum,
Environment: temperature 22 ±3 °C relative humidity 30 - 72 %
artificial light 6.00 a.m. - 6.00 p.m.
Route of administration:
oral: unspecified
Vehicle:
deionised water
Details on exposure:
On the day of the experiment, the test item was formulated in deionised water. All animals received a single standard volume of 10 mL/kg body weight orally.
Duration of treatment / exposure:
24 h and 48 h
Frequency of treatment:
1
Post exposure period:
24 h and 48 h
Remarks:
Doses / Concentrations:
24 h preparation interval: 500, 1000, and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw.
Basis:
no data
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Name: CPA; Cyclophosphamide;
Dissolved in: deionised water
Dosing: 40 mg/kg bw
Route and frequency of administration: orally, once
Volume administered: 10 mL/kg bw
Tissues and cell types examined:
Tissue: The marrow of the femora;
Cells: Two types of erythrocytes-polychromatic and normochromatic erychrocytes.
Details of tissue and slide preparation:
The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal 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-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
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 (8)) 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:
Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in deionised 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. 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 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 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 PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects 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 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, the test item is considered to be non-mutagenic in this micronucleus assay.

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

Additional information

Additional information from genetic toxicity in vivo:

Three in vitro tests and one in vivo test were performed of the test substance, including Ames test, chromosome aberration test, HPRT test and micronucleus assay.

The Ames study was performed to investigate the potential of the test item to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate.

No toxic effects, evident as a reduction in the number of revertants, 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 the test item 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.

In a second in vitro experiment ( mammalian cell gene mutation assay, HPRT locus), V79 cells cultured in vitro were exposed to FAT 40825/B TE at concentrations of

- 10, 25, 50, 100, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment I)

- 50, 100, 250, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000 and 5000 µg/mL (with metabolic activation, Experiment I)

- 25, 50, 100, 250, 500, 1000, 2000, 3500 and 5000 µg/mL (without metabolic activation, Experiment II)

- 200, 400, 800, 1400, 2000, 2600, 3200, 3800, 4400 and 5000 µg/mL (with metabolic activation, Experiment II).

FAT 40825/B TE was tested up to cytotoxic concentrations.

Biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I without metabolic activation the relative growth was 73.2% for the highest concentration (5000 µg/mL) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 17.9%. In experiment II without metabolic activation the relative growth was 28.7% for the highest concentration (5000 µg/mL) evaluated. The highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.3%.

In experiment I without metabolic activation the highest mutation rate (compared to the solvent control values) of 2.46 was found at a concentration of 5000 µg/mL with a relative growth of 73.2%.

In experiment I with metabolic activation the highest mutation rate (compared to the solvent control values) of 2.12 was found at a concentration of 3500 µg/mL with a relative growth of 54.6%.
In experiment II without metabolic activation the highest mutation rate (compared to the solvent control values) of 1.64 was found at a concentration of 2000 µg/mL with a relative growth of 54.2%.
In experiment II with metabolic activation the highest mutation rate (compared to the solvent control values) of 1.75 was found at a concentration of 5000 µg/mL with a relative growth of 34.3%.

The positive controls did induce the appropriate response.

There was no evidence of a concentration related positive response of induced mutant colonies over background.

 

In a third in vitro experiment the test item, suspended (pre-experiment and) or dissolved (experiments IB and II) 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 at least 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 respect to the current OECD Guideline 473. Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

In the absence of S9 mix and in Experiment IB and II, in the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In contrast, in, in the presence of S9 mix and in Experiment II, in the absence of S9 mix, at preparation interval 28 hrs, cytotoxicity was observed at the highest scorable concentrations. In the absence of S9 mix, and in Experiment IB and II in the presence 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.

In contrast, in the presence of S9 mix, a statistically significant and biologically relevant increase (6.0 % aberrant cells, excluding gaps) was observed at the highest scored concentration. The number of aberrant cells showed a dose-related increase, with the two highest concentrations exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps). This observation was not verified in the confirmatory Experiment IB. In Experiment II, in the absence of S9 mix, at preparation interval 18 hrs, two statistically significant increases in the number of aberrant cells (both 5.5 %) exceeding our historical control data range (0.0 - 4.0 % aberrant cells, excluding gaps) were observed. In addition, at preparation interval 28 hrs, two statistically significant and biologically relevant increases in the number of aberrant cells, excluding gaps (9.0 and 10.5 %, respectively) were observed at the scored concentrations. No relevant 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 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 as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line)in vitro.Therefore, the test item is considered to be clastogenic in this chromosome aberration test in the absence of S9 mix.

The in vivo micronucleus study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in deionised 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. 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 was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects 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 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, the test item is considered to be non-mutagenic in this micronucleus assay.

The in vivo test indicated the negative result although the in vitro chromosome aberration test indicated a positive results. The in vivo test result is more reliable for its more similarity to the human metabolic environment. In addition, the Ames test and the HPRT test showed the negative results as well. Thus, we can conclude that the test substance indicate no genetic toxicity under the experiment condition.


Justification for selection of genetic toxicity endpoint
in vivo test considered more reliable

Justification for classification or non-classification

Based on the outcome of three in vitro and one in vivo mutagenicity experiment, it is concluded that the substance FAT 40825 is not mutagenic and thus does not require classification for mutagenicity according to CLP (Regulation EC No 1272/2008) or DSD (Directive 67/548/EEC).

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