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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Eschericchia coli strain WP2 uvrA, according to the OECD guideline 471. The assay was performed in two independent experiments both with and without microsomal activation. Each concentration and the controls were 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 S9 mix in both experiments. 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 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. Based on the study results, it can be stated that during the described mutagenicity test and under the experimental conditions reported, FAT 66042/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

The substance is a member of the Stilbene Fluoorescent Whitening Agent category, group 2. Within the whole category ten over fourteen registered substances covering at least one member per group/subgroup (see data matrix in the Category Justification Report attached to the section 13 of the dossier) was tested for bacteria reverse mutation and chromosomal aberration and none of the existing tests arisen any concern for mutagenicity or genotoxicity . Furthermore, all substances of the category were modelled with OECD Toolbox and the provisional results about mutagenicity alerts were calculated for all members and their metabolites. The same alert was reported based on the H-acceptor-path3-H-acceptor. This alert explores the possibility that a chemical interacts with DNA and/or proteins via non-covalent binding, such as DNA intercalation or groove-binding (Snyder et al. 2006). Among the descriptors potentially accounting for non-covalent interactions, the present molecular framework representing two bonded atoms connecting two H bond acceptors (calculated with software Leadscope Enteprise 2.4.15-6) resulted in an increased sensitivity/specificity for what concerns the Micronucleus training set. Experimental tests both in vivo and in vitro demonstrate that this alert is not expressed in none of the substances of the group. Based on all those considerations the available studies on the analogous substances are representative for the substance under registration also that can then be considered as not genotoxic. Read across within the same group is well justified in this case also taking into account the impurities of the considered substances, since the identified organic impurities can have different substitution on the molecule, but the functional reactive groups are potentially the same and read across is justified.

Justification for selection of genetic toxicity endpoint

Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 April 2008 to 25 August 2008.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted July 21, 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 0582900
- Expiration date of the lot/batch: 25-FEB-2013
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
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):
TA 1537, genotype his C 3076; rfa-; uvrB; frame shift mutations.
TA 98, genotype his D 3052; rfa-; uvrB; R-factor; frame shift mutations.
TA 1535, genotype his G 46; rfa-; uvrB; base-pair mutations.
TA 100, genotype his G 46; rfa-; uvrB; R-factor; base-pair mutations.
WP2, genotype trp; trp; uvrA; base-pair substitutions and others
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
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:
On the day of the experiment, the test item was dissolved in deionised water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria. No precipitation of the test item occurred up to the highest investigated dose.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without metabolic activation: TA 1535, TA 100
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Without metabolic activation: TA 1537, TA 98
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation: WP2 uvrA
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With metabolic activation: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
Details on test system and experimental conditions:
PRECULTURE
From the thawed ampoules of the strains 0.5 ml suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98 and TA 100.
This nutrient medium contains per litre:
8 9 Merck Nutrient Broth (MERCK, 0-64293 Darmstadt)
5 9 NaCI (MERCK, 0-64293 Darmstadt)
The bacterial cultures were incubated in a shaking water bath for 4 hours at 37 °C. The optical density of the bacteria was determined by absorption measurement and the obtained values indicated that the bacteria were harvested at the late exponential or early stationary phase (10E8-10E9 cells/ml).

Selective Agar: the plates with the selective agar were obtained from E. Merck, 0-64293 Darmstadt.
Overlay Agar: the overlay agar contains per litre:
for Salmonella strains:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
10.5 mg L-Histidine x HCl x H2O*
12.2 mg Biotin*
For Escherichia coli:
6.0 g MERCK Agar Agar*
6.0 g NaCI*
2.5 mg Tryptophan*
* (MERCK, 0-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.

S9 PREPARATION
Phenobarbital/β-Naphthoflavone induced rat liver S9 is used as the metabolic activation system. The S9 is prepared from 8 - 12 weeks old maleWistar Hanlbm rats, weight approx. 220-320 g induced by applications of 80 mg/kg b.w. Phenobarbital Lp. (Oesitin; 0-22335 Hamburg) and β-Naphthoflavone p.o. (Aldrich, 0-89555 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 KCI 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 31.4 mg/ml. 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 KCI
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix was stored in an ice bath.

PRE-EXPERIMENT TOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

DOSE SELECTION
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since-no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration.

TEST PERFORMANCE
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl test solution at each dose level (solvent or reference mutagen solution (positive control))
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation)
100 µl Bacteria suspension (cf. test system, pre-culture of the strains)
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution (solvent or reference mutagen solution (positive control)), 500 µl S9 mix / S9 mix substitution buffer and 100 µl bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After preincubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of 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.
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA
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 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. They showed a distinct increase of induced revertant colonies.
Remarks on result:
other: all strains/cell types tested

Summary of Results Pre-Experiment and Experiment I

Metabolic Activation Test-Group Dose level (µg/plate) Revertant Colony Counts (Mean ± SD)
TA1535 TA1537 TA98 TA100 WP2 uvr/A

Without activation

Deionised water 27 ± 7 9 ± 0 32 ± 3 132 ± 5 46 ± 5
Untreated 36 ± 9 9 ± 1 29 ± 6 124 ± 7 43 ± 2
Test item 3 µg 38 ± 3 8 ± 1 25 ± 5 125 ± 2 55 ± 6
10 µg 31 ± 6 12 ± 4 34 ± 8 129 ± 13 55 ± 11
      33 µg 39 ± 10 12 ± 2 32 ± 13 135 ± 10 48 ± 4
100 µg 40 ± 0 9 ± 1 33 ± 6 149 ± 11 51 ± 12
333 µg 32 ± 7 14 ± 2 28 ± 6 140 ± 10 53 ± 7
1000 µg 25 ± 1 9 ± 1 31 ± 4 136 ± 10 49 ± 2
2500 µg 32 ± 7 9 ± 2 32 ± 4 124 ± 16 49 ± 7
5000 µg 32 ± 3 12 ± 2 29 ± 8 132 ± 16 55 ± 3
NaN3 10 µg 3129 ± 34 3103 ± 85
4-NOPD 10 µg 367 ± 15
4-NOPD 50 µg 68 ± 16
MMS 3.0 µl 1217 ± 46

With activation

Deionised water 32 ± 7 14 ± 2 37 ± 18 144 ± 19 85 ± 1
Untreated 29 ± 2 14 ± 2 40 ± 3 138 ± 10 72 ± 11
Test item 3 µg 26 ± 4 14 ± 2 34 ± 2 144 ± 16 67 ± 4
10 µg 35 ± 13 13 ± 3 32 ± 7 150 ± 5 68 ± 4
33 µg 36 ± 8 11 ± 3 40 ± 5 139 ± 13 58 ± 12
100 µg 34 ± 8 13 ± 3 36 ± 6 147 ± 11 57 ± 6
333 µg 33 ± 12 13 ± 7 29 ± 2 141 ± 13 62 ± 13
1000 µg 27 ± 3 20 ± 10 29 ± 5 138 ± 8 60 ± 2
2500 µg 27 ± 5 14 ± 7 33 ± 5 142 ± 8 65 ± 8
5000 µg 21 ± 1 10 ± 2 32 ± 1 148 ± 4 66 ± 20
2-AA 2.5 µg 298 ± 29 203 ± 13 1522 ± 58 1968 ± 159
2-AA 10.0 µg 203 ± 27

NaN3: sodium azide

2 -AA: 2-aminoanthracene

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

MMS: methyl methane sulfonate

Conclusions:
FAT 66042/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and the Eschericchia coli strain WP2 uvrA, according to the OECD guideline 471. The assay was performed in two independent experiments both with and without microsomal activation. Each concentration and the controls were 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 S9 mix in both experiments. 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 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. Based on the study results, it can be stated that during the described mutagenicity test and under the experimental conditions reported, FAT 66042/A did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

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

Genetic toxicity in vivo

Description of key information

no data available

Additional information

Justification for classification or non-classification

Based on the findings of the genetic toxicity studies, the test substance does not considered to be classified according to the EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.