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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The target substance FAT 40032 has been investigated for mutagenic effects in Salmonella typhimurium reverse mutation assay and in an in vitro HPRT test on mammalian cells which both showed negative results.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
not applicable
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
bacterial forward mutation assay
Specific details on test material used for the study:
Name: FAT 40032/E CIBACRON BRAUN P-6R ROHFEUCHT (Laborgetrocknet)
Batch No.: Op. 11
CAS No.: 70161-16-9
Aggregate State at RT: solid
Colour: brown
Purity: 75.7 % active ingredient
Target gene:
None
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver microsomal activation (S9 mix)
Test concentrations with justification for top dose:
33.3; 100.0; 333.3; 1000.0; 2500.0 and 5000.0 ug/plate.
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
The strains are derived from S. typhimurium strain LT2 and due to a mutation in the histidine locus are histidine dependent. Additionally, due to the "deep rough" (rfa-minus) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation causes a reduction in the activity of an excision repair system. The latter alteration includes mutational processes in the nitrate reductase and biotin genes produced in a UV-sensitive area of the gene named "uvrB-minus". In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries the ampicilline resistance marker. The strain TA 102 does not contain the uvrB-mutation. Additionally, TA 102 contains the multicopy plasmid pAQl, which carries the hisG428 mutation and a tetracycline resistance gene. TA 102 contains the ochre mutation in hisG gene.

Regular checking of the properties of the strains with regard to membrane permeability, ampicilline- and tetracycline-resistance as well as normal spontaneous mutation rates is performed in CCR according to Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains were obtained from Dr. Heinz Träger, Knoll AG, D-6700 Ludwigshafen, F.R.G.

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO in liquid nitrogen.

Pre-cultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. This nutrient medium contains per litre:

8 g Merck Nutrient Broth
5 g NaCl

The bacterial culture was incubated in a shaking water bath for 10 hours at 37 °C.

Selective Agar
2.0 % Vogel-Bonner-Glucose-Minimal-Agar was used as selective agar. Each petri dish was filled with 20 ml of this nutrient medium. Sterilisations were performed at 121 °C in an autoclave.

Overlay Agar
The overlay agar contains per litre:
6.0 g Merck Agar Agar
6.0 g NaCl
10.5 mg L-histidine x HCl x H20
12.2 mg biotin

Sterilisations were performed at 121 °C in an autoclave.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
S9 (Preparation by C C R)

The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, strain WU (SAVO-Ivanovas, med.
Versuchstierzuchten GmbH, D-7964 Kisslegg, F.R.G.; weight approx. 150 - 200 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-7500 Karlsruhe, F.R.G.) in olive oil 5 days previously.

After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised. The homogenate, diluted 1 + 3 in KCl was centrifuged cold at 9,000 g for 10 minutes. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -7 0° C. Small numbers of the ampoules are kept at -20 °C for only several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-8000 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).

The protein concentration in the S9 preparation was 33.6 mg/ml (lot 240292) and 31.6 mg/ml (lot 060792)
Rationale for test conditions:
None
Evaluation criteria:
None
Statistics:
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates - normal range of spontaneous reversion rates. Due to international guidelines a statistical evaluation of the results is recommended. However, no evaluated statistical procedure can be recommended for analysis of data from the bacterial
assays at this time. A test article is considered as positive if either a dose related and reproducible increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced. A test article producing neither a dose related and reproducible increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system.

A significant response is described as follows: A test article is considered as mutagenic if in strain TA 100 and TA 102 the number of reversions is at least twice as high and in strains TA 1535, TA 1537, and TA 98 it is at least three times higher as compared to the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the above described enhancement factors or not.

BIOMETRY:
No appropriate statistical method is available.
Key result
Species / strain:
other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Toxic effects, evidenced by a reduction in the number of revertants, occurred only in strain TA 100 in experiment II (preincubation test) without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40032/E at any dose level, either in the presence or 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 significance. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

None

Conclusions:
FAT 40032/E is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.
Executive summary:

A study was performed to investigate the potential of FAT 40032/E to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500 and 5000 µg/plate. Toxic effects, evidenced by a reduction in the number of revertants, occurred only in strain TA 100 in experiment II (preincubation test) without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40032/E at any dose level, either in the presence or 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 significance. 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 article did not induce point mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, FAT 40032/E is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
None
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
None
Target gene:
The purpose of this study is to evaluate a test article for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) in Chinese hamster ovary (CHO) cells, as assayed by colony growth in the presence of 6 thioguanine (TG resistance, TGr)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Arcolor 1254-induced rat liver S9
Test concentrations with justification for top dose:
In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL
In the definitive mutagenicity assay, the concentrations tested were 125, 250, 500, 1000 and 2000 µg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 µg/mL without S9
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water (test article); DMSO (Ethylmethanesulfonate, Benzo(a)pyrene)
- Justification for choice of solvent/vehicle: Water was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: single cultures in the preliminary toxicity assay; duplicate cultures in the mutagenicity assay

NUMBER OF CELLS EVALUATED: 2.4 x 10E6 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: adjusted relative survival
Rationale for test conditions:
None
Evaluation criteria:
The test substance was considered to have produced a positive response if it induced a dose-related increase in mutant frequency and an increase exceeding the 95 % historical vehicle control limits in at least one test dose level as compared with the concurrent vehicle control (p<0.01). If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director used sound scientific judgment and clearly reported and described any such considerations.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Preliminary toxicity assay: The pH of the cultures was adjusted at all concentrations with and without S9 to maintain neutral pH; Definitive mutagenicity assay: The test substance did not have an adverse impact on the pH of the cultures (pH 7.29 at the top dose).
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120 % in the preliminary toxicity assay.
- Precipitation: No visible precipitate was observed at the beginning or end of treatment in both the assays.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Under the conditions of the assay described in this report, FAT 40032/G was concluded to be negative for the induction of forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, in the in vitro mammalian cell forward gene mutation (CHO/HPRT) assay.
Executive summary:

The test substance, FAT 40032/G, was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). Water was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12 % at a concentration of 2000 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 µg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 µg/mL without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36% at a concentration of 2000 µg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 µg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). All control data were within the 95% control limits except for the solvent control mutant frequency data with S9. However, these values were acceptable as they were within the observed historical control range. The positive controls induced significant increases in mutant frequency (p < 0.01).

All positive and vehicle control values were within acceptable ranges except the condition mentioned above, and all criteria for a valid assay were met.

These results indicate FAT 40032/G was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

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

Genetic toxicity in vivo

Description of key information

An in vivo genetic toxicity study was performed to investigate the potential of FAT 40032 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The substance was regarded not to be clastogenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genetic toxicity database of FAT 40032 consists of a bacterial reverse mutation assay and an in vitro mammalian cell gene mutation assay. However, to complete data requirement at this tonnage level as well as genetic toxicity assessment of the target substance, a study investigating clastogenic potential is required. The genetic toxicity database of read across substance Reactive Black 039 (FAT 40171: pentasodium salt and FAT 45168 lithium sodium salt) consists of a bacterial reverse mutation assays (with both salts), an in vitro mammalian cell gene mutation assay with pentasodium salt and an in vitro chromosomal aberration assay with lithium sodium salt. Hence the studies with the read across substance were used to complete the genetic toxicity assessment of the target substance.

Bacterial reverse mutation assay with Reactive Brown 011 (FAT 40032)

A bacterial reverse mutation assay was performed to investigate the potential of Reactive Brown 011 to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100; 333.3; 1000; 2500 and 5000 µg/plate. Toxic effects, evidenced by a reduction in the number of revertants, occurred only in strain TA 100 in experiment II (preincubation test) without metabolic activation at the highest investigated dose. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with FAT 40032/E at any dose level, either in the presence or 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 significance. Therefore, Reactive Brown 011 was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Bacterial reverse mutation assay with REACTIVE BLACK 039 (read across) (FAT 40171)

The read across substance was evaluated for the potential to induce gene mutations according to OECD Guideline 471 and EU Method B.14 in compliance with GLP. The assay was performed in two independent experiments, both with and without liver metabolic activation. Experiment I was performed as a plate incorporation assay and Experiment II as a pre-incubation assay using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Each concentration, including the controls, was tested in triplicate. The substance was tested up to 5000 µg/plate. A slight toxic effect, evidenced by a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at the highest concentration in Experiment I. The plates incubated with the test substance showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used. No substantial increases in revertant colony numbers of any of the five tester strains were observed at any dose level. Hence Reactive Black 039 was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay. 

The lithium sodium salt of REactive Black 039 (FAT 45168) was also found to be non-mutagenic in a similar bacterial reverse mutation assay.

In vitro mammalian cell gene mutation assay with Reactive Brown 011 (FAT 40032)

REactive Brown 011 was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr). In the preliminary toxicity assay, the concentrations tested were 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µg/mL. The maximum concentration evaluated was the limit dose for this assay. No visible precipitate was observed at the beginning or end of treatment. Adjusted relative survival was 96.59 and 2.12 % at a concentration of 2000 µg/mL with and without S9, respectively. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 125, 250, 500, 1000 and 2000 µg/mL with S9 and 125, 250, 500, 1000, 1200, 1500, 1800 and 2000 µg/mL without S9.

In the definitive mutagenicity assay, no visible precipitate was observed at the beginning or end of treatment. The average adjusted relative survival was 90.74 and 95.36 % at a concentration of 2000 µg/mL with and without S9, respectively. Cultures treated at concentrations of 125, 250, 500, 1000 and 2000 µg/mL with and without S9 were chosen for mutant selection. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed at any concentration evaluated with or without S9 (p > 0.05). These results indicate Reactive Brown 011 was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system.

In vitro mammalian cell gene mutation assay with Reactive Black 039 or FAT 40171 (read across)

Read across substance, Reactive Black 039 was evaluated in a mammalian cell gene mutation assay (HPRT locus). In this assay, V79 cells cultured in vitro were exposed to the substance at concentrations of 5, 10, 25, 50, 100, 250, 500, 1000, 2500 and 5000 µg/mL in the Experiment I (with and without metabolic activation); at 100, 250, 500, 750, 1000, 2000, 3000, 4000 and 5000 µg/mL (without metabolic activation, Experiment II) and at 45, 90, 180, 375, 750, 1500, 3000, 4000 and 5000 µg/mL (with metabolic activation, Experiment II). Biologically relevant growth inhibition was observed in experiment I with and without metabolic activation and in experiment II with metabolic activation. No biologically relevant growth inhibition was observed in experiment II without metabolic activation. In experiment I without metabolic activation the relative growth was 27.1 % 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 21.1 %. In experiment II without metabolic activation the relative growth was 72.5 % for the highest concentration (5000 µg/mL) evaluated. In experiment II with metabolic activation the highest concentration evaluated with metabolic activation was 5000 µg/mL with a relative growth of 34.9 %. In experiment I without metabolic activation the highest mutation rate (compared to the negative control values) of 1.37 was found at a concentration of 500 µg/mL with a relative growth of 76.2 %. In experiment I with metabolic activation the highest mutation rate (compared to the negative control values) of 2.04 was found at a concentration of 2500 µg/mL with a relative growth of 54.1 %. In experiment II without metabolic activation the highest mutation rate (compared to the negative control values) of 0.74 was found at a concentration of 1000 µg/mL with a relative growth of 77.5%. In experiment II with metabolic activation the highest mutation rate (compared to the negative control values) of 2.88 was found at a concentration of 4000 µg/mL with a relative growth of 52.4 %. There was no evidence of a concentration related positive response of induced mutant colonies over background. In conclusion, read across substance, Reactive Black 039 is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

In vitro chromosomal aberration assay with Reactive Black 039 or FAT 45168 (read across)

The clastogenic potential of the read across substance, Reactive Black 039 lithium sodium salt was evaluated in a GLP-compliant chromosome aberration test, conducted according to OECD guideline 473. In this study, Chinese hamster V79 cells were exposed to the test substance with and without metabolic activation. In cytogenetic experiment I, in the absence of S9 mix cultures after treatment with 1000 µg/mL (18 and 28 hours) as highest concentration were evaluated for cytogenetic damage; in experiment II, 2000 µg/mL (18 hours) and 300 µg/mL (28 hours) as highest concentrations were scorable. In the presence of S9 mix, in both experiments cultures treated with 5000 µg/mL as top concentration could be evaluated at preparation interval 18 hours. At preparation interval 28 hours, in experiment I 5000 µg/mL and in experiment II 2500 µg/mL were evaluated. The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible due to toxic effects (reduced cell density and/or low metaphase number, partially combined with poor metaphase quality). No biologically relevant increase in the number of chromosome aberrations was observed. It was therefore concluded that under the given experimental conditions the test substance did not induce structural chromosomal aberrations.

Based on the negative results in bacterial reverse mutation assay and the in vitro mammalian cell gene mutation assay with target substance as well as bacterial reverse mutation assays, an in vitro mammalian cell gene mutation assay and an in vitro chromosomal aberrations assay with read across substance, the target substance Reactive Brown 011 can be considered to be non-mutagenic as well as non-clastogenic. Hence, it can be considered to be devoid of genetic toxicity potential.

Justification for classification or non-classification

Based on the above discussion, FAT 40032 was considered to be not genotoxic, and hence does not warrant classification as per the CLP (Regulation EC 1272/2008) criteria.