Registration Dossier
Registration Dossier
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EC number: 263-319-6 | CAS number: 61916-41-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- disregarded due to major methodological deficiencies
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: see remarks.
- Remarks:
- Study conducted according to internationally accepted testing guidelines and performed according to GLP. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted May 26, 1983
- Deviations:
- yes
- Remarks:
- The bacterial cultures were incubated in a shaking water bath for 8 hours at 37° C. Reason for the alteration: updating.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- dated December 29, 1992
- Deviations:
- yes
- Remarks:
- The bacterial cultures were incubated in a shaking water bath for 8 hours at 37° C. Reason for the alteration: updating.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate
- Vehicle / solvent:
- - Vehicle/solvent used: on the day of experiment, the test article was dissolved in aqua bidest. The solvent was chosen because of its solubility properties. The test article did not precipitate in the overlay agar up to 5000.0 µg/plate.
- Untreated negative controls:
- yes
- Remarks:
- Concurrent untreated.
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- Without metabolic activation, strains: TA 1535 and TA 100.
- Positive control substance:
- sodium azide
- Remarks:
- Dissolved in aqua dest.
- Untreated negative controls:
- yes
- Remarks:
- Concurrent untreated.
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- Without metabolic activation, strains: TA 1537 and TA 98.
- Positive control substance:
- other: 4-nitro-o-phenylene-diamine, 4-NOPD
- Remarks:
- Dissolved in DMSO.
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- With metabolic activation.
- Positive control substance:
- other: 2-aminoanthracene, 2-AA
- Remarks:
- Dissolved in DMSO.
- Details on test system and experimental conditions:
- STORAGE
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.
PRECULTUREAS
From the thawed ampoules of the strains 0.5 ml suspension was transferred to 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin was added to the strains TA 98 and TA 100. This nutrient medium contains per litre: 8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt); 5 g NaCl (MERCK, D-64293 Darmstadt).
The bacterial cultures were incubated in a shaking water bath for 8 hours at 37 °C.
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.
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5 mg L-HistidinxHClxH20*
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)
Sterilisations were performed at 121 °C in an autoclave.
MAMMALIAN MICROSOMAL FRACTION S9 MIX
- S9 preparation
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats, strain Hanlbm (BRL, CH-4414 Füllinsdorf weight approx. 220 - 320 g) which received a single i.p. injection of 500 mg/kg b.w. Aroclor 1254 (Antechnika, D-76275 Ettlingen, F.R.G.) in olive oil 5 days previously.
After cervical dislocation the livers of the animals were re-moved, washed in 150 mM KCl and homogenised. The homogenate was diluted 1+3 in KCl and centrifuged at 9000 g for 10 minutes at 4 °C. A stock of the supernatant containing the microsomes was frozen in ampoules of 2, 3 or 5 ml and stored at -80 °C. Small numbers of the ampoules are kept at -20 °C for up to several weeks before use. The standardisation of the protein content was made using the analysis kit of Bio-Rad Laboratories, D-80939 München: Bio-Rad protein assay, Catalogue 500 000 6 (6).
The protein concentration in the S9 preparation was 30.6 mg/ml (lot 250795).
- 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 15% v/v. The composition of the co-factor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-ortho-phosphate-buffer, pH 7.4.
During the experiment the S9 mix will be stored in an ice bath.
PRE-EXPERIMENTAL FOR TOXICITY
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with 3 plates each. Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
According to the results of this pre-experiment the concentrations applied in the main experiments were chosen.
EXPERIMENTAL PERFORMANCE
For each strain and dose level, including the controls three plates were used as a minimum.
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 (negative control) 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
After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.
NUMBER OF REPLICATIONS
The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. - Evaluation criteria:
- 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.
Range of spontaneous reversion frequencies:
TA 1535: 10-29
TA 1537: 5-28
TA 98: 15-37
TA 100: 77-189 - Statistics:
- 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 significant response is described as follows:
A test article is considered mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 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. - Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- other: test article induced gene mutations by frameshifts in the genome of the strain TA 1537 and shows a possible mutagenic potential in the strain TA 98.
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Remarks:
- No toxic effects, evidenced by a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000.0 µg/plate.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- No toxic effects, evidenced by a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000.0 µg/plate.
The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used.
Substantial, dose dependent increases in revertant colony numbers were observed in the strain TA 1537 following treatment with test item in the absence and presence of metabolic activation (S9 mix) in both experiments.
The enhancement factor of three was already exceeded at a concentration of 333 µg/plate in the first experiment. In the second experiment this limit was reached at 333 µg/plate in the absence of metabolic activation. In the presence of metabolic activation the numbers of revertant colonies were lower and the factor of three was exceeded at the highest concentration only.
Slight, but reversible and concentration dependent increases in revertant colony numbers with and without S9 occurred in the strain TA 98 as well. Since the enhancement factors did not quite reach the limit of three a possible mutagenic potential of the test substance is indicated in this strain.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. - Remarks on result:
- other: the substance has nitro groups within the structure, therefore the standard strains in the Ames test are not diagnostic to detect the mutagenicity
- Conclusions:
- Interpretation of results (migrated information):
positive
The substance is considered to be mutagenic in Salmonella typhimurium reverse mutation assay. - Executive summary:
Method
This study was performed to investigate the potential of the substance to induce gene mutations according to the plate incorporation test (experiments I and II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, according to the OECD guideline 471.
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; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.
Results
No toxic effects, evident as a reduction in the number of revertants, occurred in any of the test groups with and without metabolic activation up to 5000.0 µg/plate. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Substantial, dose dependent increases in revertant colony numbers were observed in the strain TA 1537 following treatment with test substance in the absence and presence of metabolic activation (S9 mix) in both experiments. Slight, but reproducible and dose dependent increases in revertant colony numbers also occurred in strain TA 98 with and without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Conclusion
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did induce gene mutations by frameshifts in the genome of the strain TA 1537 and shows a possible mutagenic potential in the strain TA 98. Therefore, the substance is considered to be mutagenic in the current Salmonella typhimurium reverse mutation assay.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Study period:
- December, 1984
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Justification for type of information:
- Study performed according to the GLP Principles. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO).
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI).
A nutrient agar (0.8 % Difco nutrient broth, 1.5 % Difco agar, 0.5 % NaCI) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Plates were then transferred to the refrigerator, for up to one week.
CHECKING OUT TESTER STRAINS
All strains were tested for the presence of their mutations.
a)
_The mutation in the histidine operon, basic to the test system, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
_Bacteria of the nutrient agar plate were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
_The plates were incubated at 37 °C overnight. Growth, for all strains, was seen only where histidine and biotin were present.
b)
Sensitivity to crystal violet is a check for the presence of the deep rough ( rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c)
Two of the tested strains (TA 98 and TA 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the plasmid, a sterile filter paper disc containing ampicillin (10 µl of 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA 1535 and TA 1537 a zone of inhibition occurs around the disc, but for TA 98 and TA 100 where the R factor is present, no zone of inhibition is seen.
d)
The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a nutrient agar plate. One half of the plate was irradiated for 20 seconds under a 15 watt UV lamp at a distance of approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.
INDUCTION OF RAT LIVER ENZYMES
Three male Sprague-Dawley rats weighing approximately 200 grams were given an i.p. injection of Arocior 1254 (IVIonsanto) in peanut oil at a dose of 500 mg/kg.
Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenized in three times their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant (termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70 °C. - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver enzymes
- Test concentrations with justification for top dose:
- 20, 80, 320, 1280 and 5120 µg/plate
- Vehicle / solvent:
- dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- other: 2-anthramine (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and daunomycidine (DM)
- Remarks:
- Without S9: TA 1535 and TA 100 MNNG (1.6 µg); TA 1537 9-AA(50 µg); TA 98 DM (5 µg). With S9: all strains 2-AA (12.5 µg)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose.
The top agar is a 0.6 % Bacto-Difco agar, 0.5 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C.
Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water.
The appropriate dilutions were made from a 51.2 mg/stock.
The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.
The test system was as follows; the under mentioned compounds were added to sterile glass tubes in the given order:
- 0.1 ml of solution of compound to be tested
- 0.5 ml of S-9 mix (or 0.85 % saline)
- 0.1 ml of bacterial suspension (2-3 x 10^8 bacteria)
- 2 ml of molten top agar at 45 °C.
The tubes were agitated using a vortex mixer and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark. The colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony Counter. Each strain has a characteristic spontaneous reversion rate.
An examination under the microscope was used to verify the presence of the background produced by growth of auxotrophic bacteria on traces of histidine and biotin, or the absence of background due to a toxic effect of the test substance.
NUMBER OF REPLICATIONS
Every concentration was tested in triplicate. - Evaluation criteria:
- The criteria of mutagenicity used are a doubling of the spontaneous reversion rate and a dose-effect relationship.
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- a toxic effect was observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- a toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Additional information on results:
- Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.
With strain TA 98 the evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 1280 pg with S-9 mix, at 80 and 320 µg without S-9 mix. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.
A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive
A mutagenic effect was observed with the strain TA 98 (with and without metabolic activation). - Executive summary:
The substance was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation.
Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.
With strain TA 98 the evidence for mutagenicity existed both in the absence and presence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 1280 µg with S-9 mix, at 80 and 320 µg without S-9 mix. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.
A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.
Conclusion
From the results of Ames test, it appears that the test substance presents only a very slight mutagenic effect in this system. This effect is observed only with one tester strain (TA 98). This effect is markedly diminished when liver microsomial enzymes are added to the preparation suggesting that the compound might be detoxified in vivo.
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Study period:
- January, 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Justification for type of information:
- Study performed according to the GLP Principles. The OECD recommended combination of strains was not respected: none of the E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 was tested.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Details on mammalian cell type (if applicable):
- GROWING AND CONSERVATION OF BACTERIAL TEST STRAINS
The bacterial strains were kept as frozen broth cultures, in aliquots of 0.5 ml at -70 °C with 8.0 % dimethylsulfoxide (DMSO).
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (normal Difco nutrient broth for strains TA 1535 and TA 100, and double strength Difco nutrient broth for TA 1537 and TA 98, in 0.5 % NaCI).
A nutrient agar (0.8 % Difco nutrient broth, 1.5 % Difco agar, 0.5 % NaCI) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37 °C for 16 hours, while the plate was incubated at 37 °C overnight. Plates were then transferred to the refrigerator, for up to one week.
CHECKING OUT TESTER STRAINS
All strains were tested for the presence of their mutations.
a)
_The mutation in the histidine operon, basic to the test system, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
_Bacteria of the nutrient agar plate were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
_The plates were incubated at 37 °C overnight. Growth, for all strains, was seen only where histidine and biotin were present.
b)
Sensitivity to crystal violet is a check for the presence of the deep rough ( rfa) mutation, the loss of the lipopolysaccharide coat on the bacterial surface.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this surface. A zone of inhibition around the disc, after 24 hours incubation, shows the presence of the (rfa) mutation.
c)
Two of the tested strains (TA 98 and TA 100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the plasmid, a sterile filter paper disc containing ampicillin (10 µl of 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA 1535 and TA 1537 a zone of inhibition occurs around the disc, but for TA 98 and TA 100 where the R factor is present, no zone of inhibition is seen.
d)
The uvrB deletion, the loss of the excision repair system, makes the bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a nutrient agar plate. One half of the plate was irradiated for 20 seconds under a 15 watt UV lamp at a distance of approximately 30 cm. After 24 hours' incubation, growth was found only on the unirradiated part of the streak for all strains.
INDUCTION OF RAT LIVER ENZYMES
Three male Sprague-Dawley rats weighing approximately 200 grams were given an i.p. injection of Arocior 1254 (IVIonsanto) in peanut oil at a dose of 500 mg/kg.
Five days after the injection, the rats were anaesthetized with ether, decapitated and the livers removed in a sterile manner. The livers were homogenized in three times their volume of sterile 0.15 M KCl, pooled together and centrifuged for 15 min at 9000 xg (Beekman refrigerated ultracentrifuge). The supernatant (termed the S-9 fraction) was aliquoted into sterile tubes and frozen at -70 °C. - Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver enzymes
- Test concentrations with justification for top dose:
- 20, 80, 320, 1280 and 5120 µg/plate
- Vehicle / solvent:
- dimethylsulfoxide (DMSO)
- Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- other: 2-anthramine (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and daunomycidine (DM)
- Remarks:
- Without S9: TA 1535 and TA 100 MNNG (1.6 µg); TA 1537 9-AA(50 µg); TA 98 DM (5 µg). With S9: all strains 2-AA (12.5 µg)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
PREPARATION OF MATERIALS
The minimal-glucose agar medium base was prepared from a 1.5 % Bacto-Difco agar in Vogel & Bonner E medium with 2 % glucose.
The top agar is a 0.6 % Bacto-Difco agar, 0.5 % NaCI solution. Before use, the agar was melted in a boiling water bath and then left to equilibrate to 45 °C.
Ten ml of sterile 0.5 mM L-histidine - 0.5 mM biotin were added per 100 ml of top agar.
The compounds to be tested were prepared fresh daily in DMSO or in sterile water.
The appropriate dilutions were made from a 51.2 mg/stock.
The S-9 mix was prepared fresh daily from sterile stocks. 0.5 ml contains:
4.0 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50.0 µ moles Phosphate Buffer, pH 7.4
150.0 µl liver homogenate.
The test system was as follows; the under mentioned compounds were added to sterile glass tubes in the given order:
- 0.1 ml of solution of compound to be tested
- 0.5 ml of S-9 mix (or 0.85 % saline)
- 0.1 ml of bacterial suspension (2-3 x 10^8 bacteria)
- 2 ml of molten top agar at 45 °C.
The tubes were agitated using a vortex mixer and poured onto the minimal medium base. The plates were incubated for 48 hours at 37 °C in the dark. The colonies, the revertants to the wild type, were counted manually or electronically using a Fisher Colony Counter. Each strain has a characteristic spontaneous reversion rate.
An examination under the microscope was used to verify the presence of the background produced by growth of auxotrophic bacteria on traces of histidine and biotin, or the absence of background due to a toxic effect of the test substance.
NUMBER OF REPLICATIONS
Every concentration was tested in triplicate. - Evaluation criteria:
- The criteria of mutagenicity used are a doubling of the spontaneous reversion rate and a dose-effect relationship.
- Species / strain:
- other: S. typhimurium TA 1535, TA 1537 and TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- a toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- a toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Additional information on results:
- Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.
With the strain TA 98 the evidence for mutagenicity existed only in the absence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 320 µg without S-9 mix, at 80 µg an increased number of revertants was noted. Only a doubling of the revertant number was observed in presence of S-9 mix at 1280 µg. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.
A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results (migrated information):
positive
A mutagenic effect was observed with the strain TA 98 (with and without metabolic activation). - Executive summary:
The substance was tested with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 at concentrations from 20 to 5120 µg per Petri dish both in the presence and absence of metabolic activation.
Under the experimental conditions defined in the protocol, and employing a doubling of the spontaneous reversion rate and dose-effect relationship as criteria of mutagenicity, the product was mutagenic for Salmonella typhimurium strain TA 98. No mutagenic effect was noted with the strains TA 1535, TA 1537 and TA 100.
With the strain TA 98 the evidence for mutagenicity existed only in the absence of a liver microsomal enzyme preparation (S-9 mix) from male rats pretreated with Aroclor 1254 at 320 µg without S-9 mix, at 80 µg an increased number of revertants was noted. Only a doubling of the revertant number was observed in presence of S-9 mix at 1280 µg. A toxic effect was noted at 5120 µg with S-9 mix and at 1280 µg and 5120 µg without S-9 mix.
A toxic effect was also observed with the strains TA 1537 at 5120 µg of product in presence and absence of S-9 mix and with the strain TA 100 at 5120 µg only in absence of S-9 mix.
Conclusion
From the results of Ames test, it appears that the test substance presents only a very slight mutagenic effect in this system. This effect is observed only with one tester strain (TA 98). This effect is markedly diminished when liver microsomial enzymes are added to the preparation suggesting that the compound might be detoxified in vivo.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Study period:
- From March 31 from April 22, 1994
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 1983
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 1992
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- histidine locus
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- MAMMALIAN MICROSOMAL FRACTION S9 MIX
- Test concentrations with justification for top dose:
- Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate
Exp. II: 100.0; 333.3; 1000.0; 2500.0; 3333.3; and 5000.0 µg/plate
According to the results of the pre-experiment the concentrations applied in the main experiments were chosen. The concentration range covered two logarithmic decades. The maximum concentration was 5000.0 ug/plate (active ingredient). The concentration range included two logarithmic decades. In this study six adequately spaced concentrations were tested. Two independent experiments were performed. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMF
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity for the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- other: 4-nitro-o-phenylene-diamine
- Remarks:
- Without metabolic activation
- 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
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Experiment I and II were performed as plate incorporation assay
DURATION
- Exposure duration: at least 48 hours at 37° C in the dark
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test article a pre-experiment was performed with strains TA 98 and TA 100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test).
Toxicity of the test article can be evidenced by a reduction in the number of spontaneous revertants, a clearing of the bacterial background lawn, or by degree of survival of treated cultures. - Evaluation criteria:
- EVALUATION OF RESULTS
The generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative controls and test plates
- normal range of spontaneous reversion rates.
Range of spontaneous reversion frequencies*
1535 1537 98 100
10-29 5-28 15-57 77-189
*These values refer to the negative control without metabolic activation and represent our historical control range in 1993
A test article is considered 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 mutagenic if in strain TA 100 the number of reversions is at least twice as high and in strains TA 1535, TA 1537 and TA 98 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. - Statistics:
- 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.
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- up to 5000.0 µg/plate in experiment I and II
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Remarks:
- with S9 mix up to 2500 µg/plate in exp. I and II. Without S9 mix up to 5000.0 µg/plate in experiment I and II
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- At 5000.0 µg/plate in experiment I and at 2500.0, 3333.3 and 5000.0 µg/plate without S9 mix, as well as at 3333.3, and 5000.0 ug/plate with S9 mix in experiment II
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- The plates incubated with the test article showed normal background growth up to 5000.0 ug/plate with and without S9 mix in all strains used.
- Conclusions:
- Mutagenic
- Executive summary:
Method
This study was performed to investigate the potential of the test substance to induce gene mutations using the plate incorporation test with the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, according to the OECD guideline 471.
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:
Exp. I: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate;
Exp. II: 100.0; 333.3; 1000.0; 2500.0; 3333.3; and 5000.0 µg/plate.
Results
Toxic effects, evidenced by a reduction in the number of revertants, occurred in strain TA 1535 at 5000.0 µg/plate in experiment I and at 2500.0, 3333.3 and 5000.0 µg/plate without S9 mix, as well as at 3333.3, and 5000.0 µg/plate with S9 mix in experiment II. The plates incubated with the test article showed normal background growth up to 5000.0 µg/plate with and without S9 mix in all strains used. Dose-dependent increases in revertant colony numbers were observed following treatment with the test substance in strain TA 1537 without S9 mix and in strain TA 98 with and without S9 mix in experiment I and II. Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.
Conclusion
Mutagenic.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- other: read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Study period:
- From March to April, 1995.
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- The test was conducted by means of Read Across approach. The reliability of the source study report is 1. Further information was attached at section 13
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- Large stocks of the V79 cell line are stored in liquid nitrogen in the cell bank of testing laboratory allowing the repeated use of the same cell culture batch in experiments. Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium.
Each batch is screened for mycoplasma contamination and checked for karyotype stability and spontaneous mutant frequency. Consequently, the parameters of the experiments remain similar because of the reproducible characteristics of the cells. - Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Experiment I:
without S9 mix: 1.0; 3.0; 10.0; 15.0; 25.0 and 50.0 µg/ml
with S9 mix: 3.0, 30.0, 100.0; 150.0; 200.0 and 250.0 µg/ml
Experiment II:
without S9 mix: 1.0; 20.0; 30.0; 40.0; 45.0 and 50.0 µg/ml
with S9 mix: 3.0; 100.0; 200.0; 230.0; 240.0 and 250.0 µg/ml
Experiment III:
without S9 mix: 1.0; 5.0; 10.0; 15.0; 20.0 and 25.0 µg/ml
with S9 mix: 3.0; 50.0; 100.0; 150.0; 180.0 and 200.0 µg/ml - Vehicle / solvent:
- - Solvent used: DMSO
- Justification for choice of solvent: the solvent was chosen according to its solubility properties and its non-toxicity for the cells. The final concentration of DMSO in the culture medium did not exceed 1 % v/v. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- ethylmethanesulphonate
- Remarks:
- Without metabolic activation: EMS; with metabolic activation: DMBA.
- Details on test system and experimental conditions:
- PRE-TEST on TOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. The general culturing and experimental conditions in this pre-test were the same as described below for the mutagenicity experiment. The following method was used in the pre-test:
XTT-Assay. The XTT-assay is based on the cleavage of the yellow tetrazolium salt XTT to form an orange formazan dye by hydrogenase activity in active mitochondria. 18 - 20 h after treatment with the test article the XTT-assay was initiated by adding a mixture of XTT-labelling reagent with an electron coupling reagent (PMS). After 4 h of incubation the absorption was determined at 450 nm (690 nm reference) using an ELISA reader. The viabilities of the cells are calculated as percentages of the solvent controls and reported as tables.
DOSE SELECTION
In the pre-test of toxicity (XTT-assay) the extinction (measured at 450/690 nm) was reduced after treatment with concentrations higher than 30.0 µg/ml (without S9 mix) and slightly reduced at 100 µg/ml in DMSO (with S9 mix).
Experiment I was performed with six concentrations ranging from 1.0 to 50.0 µg/ml without and from 3.0 to 250.0 µg/ml with metabolic activation. The highest concentration (50 µg/ml) without S9 could not be assessed in this experiment due to strong toxic effects.
Strong toxic effects occurred in the second experiment already at 40 µg/ml and above without metabolic activation and at 200 µg/ml or higher with metabolic activation. In experiment III most evaluated concentrations were close to the limit of toxicity to detect possible mutagenic effects in the toxic range.
EXPERIMENTAL PERFORMANCE
Seeding
Threedays old exponentially growing stock cultures (more than 50 % confluent) were trypsinized at 37 °C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2 % in Ca-Mg-free salt solution.
The Ca-Mg-free salt solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1000 mg, NaHC03 350 mg
Prior to the trypsin treatment the cells were rinsed with Ca-Mg-free salt solution containing 200 mg/l EDTA (ethylene diamine tetraacetic acid).
The cell suspension was seeded into plastic culture flasks. Approximately 1.5x10^6 (single culture) and 5x10^2 cells (in duplicate) were seeded in MEM with 10 % FCS (complete medium) for the determination of mutation rate and toxicity, respectively.
Treatment
After 24 h the medium was replaced with serum-free medium containing the test article, either without S9 mix or with 50 µl/ml S9 mix. After 4 h this medium was replaced with complete medium after two washing steps with "saline G".
The "saline G" solution was composed as follows (per litre): NaCl 8000 mg, KCl 400 mg, Glucose 1100 mg, Na2HP04x7H20 290 mg, KH2P04 150 mg,.
pH is adjusted to 7.2.
Experimental scheme
Day 1: Subculturing of a log-phase culture which showed an initial spontaneous mutation rate at the beginning of the experiment of 3.2 (experiment I),. 11.1 (experiment II) and 4.8 (experiment 3) mutants per 10^6 cells.
a) About 500 cells in 5 ml medium/25 cm2-plastic-flask for cloning efficiency; in duplicate per experimental point
b) 1x10^6 cells in 30 ml medium/175 cm2-plastic-flask for the mutagenicity test, 1 flask per experimental point
Day 2: Treatment of a) and b)
experiment I
Day 5: Sub-culturing of b) in 175 cm2-plastic-flasks 1.5x10^6 cells in 30 ml medium/175 cm2-plastic-flasks
experiment II
Day 6: see day 5
Day 8: Fixation and staining of colonies in a)-flasks determination of concentration-related cloning efficiency
Day 9: Sub-culturing of b) in five 80 cm2-plastic-flasks containing selective medium: mutant selection (about 3-5x10^5 cells/flask);
sub-culturing of b) in two 25 cm2-flasks for cloning efficiency (about 500 cells/flask)
Day 16: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (cloning efficiency).
Day 18: Fixation and staining of colonies in b) - derived flasks seeded on day 9 (mutant selection).
The third experiment was performed according to the schedule of experiment I.
The cultures were incubated at 37 °C in a humidified atmosphere with 4.5 % CO2. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope. - Evaluation criteria:
- A test article is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response for one of the test points.
A test article producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
The test article is classified as mutagenic if it induces a reproducible mutation frequency that is at least three times higher than the spontaneous mutation frequency in the experiment at one or more of the concentrations.
The test article is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding negative control data. If there is by chance a low spontaneous mutation rate in the range normally found (0 - 45 mutants per 10^ cells) a concentration-related increase of the mutations within this range has to be discussed. - Statistics:
- Since the distribution of mutant cells does not follow known statistical models, an adequate statistical method is not available.
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Without metabolic activation strong toxic effects occurred already at concentrations as low as 25.00 µg/ml. With metabolic activation, the concentration of the test article could be increased up to 250 µg/ml in experiment I and 200 µg/ml in experiment II. Precipitation of the test article occurred at concentrations of 100 µg/ml and above.
The cloning efficiency of the cells was reduced below 30 % at the highest concentrations tested.
The number of mutant colonies was not increased compared to the frequency of spontaneous mutations at any concentration of the test article. There was also no indication of a concentration depend increase of mutant colonies.
In all experiments of this study (with and without S9 mix) the range of the negative controls was from 0.6 up to 18.7 mutants per 10^6 cells; the range of the groups treated with the test article was from 4.1 up to 18.9 mutants per 10^6 cells.
EMS (0.6 mg/ml) and DMBA (3.85 µg/ml) were used as positive controls and showed a distinct increase in induced mutant colonies. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Test substance is considered to be non-mutagenic in this HPRT assay.
- Executive summary:
Method
The study was performed to investigate the potential of test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster. The assay was performed in three independent experiments, using identical procedures, both with and without liver microsomal activation. Due to unexpectedly strong toxic effects an additional experiment was required to complete the data of experiment II. In the absence of metabolic activation the concentration range was limited by toxicity, in the presence of metabolic activation by solubility of the test article.
The test article was tested with the following concentrations:
Experiment I:
without S9 mix: 1.0; 3.0; 10.0; 15.0; 25.0 and 50.0 µg/ml
with S9 mix: 3.0, 30.0, 100.0; 150.0; 200.0 and 250.0 µg/ml
Experiment II:
without S9 mix: 1.0; 20.0; 30.0; 40.0; 45.0 and 50.0 µg/ml
with S9 mix: 3.0; 100.0; 200.0; 230.0; 240.0 and 250.0 µg/ml
Experiment III:
without S9 mix: 1.0; 5.0; 10.0; 15.0; 20.0 and 25.0 µg/ml
with S9 mix: 3.0; 50.0; 100.0; 150.0; 180.0 and 200.0 µg/ml
Precipitation of the test article occurred at concentrations of 100 µg/ml and above in all three experiments.
Results
According to the pre-test on toxicity the concentration ranges were selected to yield concentration-related toxic effects. The highest concentration produced a low level of survival and the survival at the lowest concentration was approximately in the range of the negative control.
Up to the highest investigated concentration no relevant increase in mutant colony numbers was obtained in all independent experiments. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.
Conclusion
In conclusion it can be stated that during the described mutagenicity test and under the experimental conditions reported the test article did not induce gene mutations at the HPRT locus in V79 cells.
Therefore, test substance is considered to be non-mutagenic in this HPRT assay.
Referenceopen allclose all
Pre-Experiment for Toxicity
To evaluate the toxicity of the test article a pre-study was performed with strains TA 98 and TA 100.
| Substance | Concentration per plate µg |
Revenants per plate | |||
| TA 98 | TA 100 | ||||
| - | + | - | + | ||
| Negative control | - | 14 | 13 | 160 | 173 |
| Solvent control | - | 14 | 16 | 172 | 178 |
| 4-NOPD | 10 | 88 | / | / | / |
| Sodium azide | 10 | / | / | 668 | / |
| 2-aminoanthracene | 2.5 | / | 434 | / | 936 |
| test article | 3.3 | 16 | 15 | 181 | 206 |
| 10 | 13 | 13 | 166 | 208 | |
| 33.3 | 14 | 17 | 155 | 211 | |
| 100 | 18 | 15 | 167 | 206 | |
| 333.3 | 20 | 16 | 195 | 222 | |
| 1000 | 22 | 18 | 205 | 231 | |
| 2500 | 28 | 24 | 202 | 291 | |
| 5000 | 32 | 28 | 236 | 299 | |
- = without S9 mix
+ = with S9 mix
/ = not performed
Summary of Results
without S9 mix
| Concentration µg/plate | Revertants/plate mean from three plates |
|||||||
| TA 1535 | TA 1537 | TA 98 | TA 100 | |||||
| I | II | I | II | I | II | I | II | |
| Negative control | 10 | 11 | 5 | 7 | 14 | 29 | 160 | 105 |
| Solvent control | 8 | 11 | 5 | 7 | 14 | 32 | 172 | 126 |
| Positive control* | 702 | 712 | 37 | 42 | 88 | 154 | 668 | 694 |
| 33.3 | 9 | 13 | 7 | 8 | 14 | 30 | 155 | 116 |
| 100.0 | 8 | 10 | 11 | 20 | 18 | 40 | 167 | 114 |
| 333.3 | 11 | 11 | 19 | 22 | 20 | 40 | 195 | 123 |
| 1000.0 | 9 | 11 | 54 | 43 | 22 | 53 | 205 | 129 |
| 2500.0 | 11 | 21 | 74 | 58 | 28 | 71 | 202 | 149 |
| 5000.0 | 16 | 26 | 117 | 84 | 32 | 75 | 236 | 170 |
with S9 Mix
| Concentration µg/plate | Revertants/plate mean from three plates |
|||||||
| TA 1535 | TA 1537 | TA 98 | TA 100 | |||||
| I | II | I | II | I | II | I | II | |
| Negative control | 13 | 14 | 6 | 22 | 13 | 34 | 173 | 130 |
| Solvent control | 14 | 11 | 5 | 19 | 16 | 32 | 178 | 127 |
| Positive control** | 181 | 188 | 115 | 129 | 434 | 394 | 936 | 823 |
| 33.3 | 11 | 12 | 6 | 18 | 17 | 39 | 211 | 137 |
| 100.0 | 16 | 12 | 11 | 19 | 15 | 41 | 206 | 127 |
| 333.3 | 11 | 16 | 14 | 24 | 16 | 34 | 222 | 133 |
| 1000.0 | 8 | 14 | 23 | 29 | 18 | 51 | 231 | 141 |
| 2500.0 | 13 | 20 | 50 | 40 | 24 | 62 | 291 | 166 |
| 5000.0 | 13 | 23 | 89 | 68 | 28 | 75 | 299 | 198 |
* Sodium azide (10.0 µg/plate) strains TA 1535 and TA 100; 4-Nitro-o-phenylene-diamine (10.0 µg/plate) strains TA 1537 and TA 98
**2-Aminoanthracene (2.5 µg/plate)
| With S9 | ||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | |
| Spontaneous reversion | 49 (SD = 2) | 142 (SD = 13) | 17 (SD = 3) | 9 (SD = 2) |
| Control DMSO | 34 (SD = 5) | 121 (SD = 5) | 14 (SD = 5) | 12 (SD = 2) |
| Positive control | 2497 (SD = 437) | 2613 (SD = 78) | 817 (SD = 52) | 275 (SD = 26) |
| Test substance (µg) | ||||
| 20.0 | 57 (SD = 7) | 117 (SD = 11) | 13 (SD = 3) | 11 (SD = 5) |
| 80.0 | 59 (SD = 7) | 126 (SD = 11) | 12 (SD = 3) | 9 (SD = 1) |
| 320 | 77 (SD = 7) | 135 (SD = 4) | 10 (SD = 2) | 10 (SD = 6) |
| 1280.0 | 118 (SD = 22) | 115 (SD = 9) | 12 (SD = 1) | 16 (SD = 4) |
| 5120.0 | <0> | 174 (SD = 1) | 28 (SD = 3) | <0> |
| Without S9 | ||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | |
| Spontaneous reversion | 21 (SD = 3) | 121 (SD = 10) | 24 (SD = 2) | 6 (SD = 2) |
| Control DMSO | 21 (SD = 5) | 100 (SD = 1) | 18 (SD = 6) | 4 (SD = 1) |
| Positive control | 863 (SD = 106) | 2088 (SD = 62) | 2190 (SD = 46) | 233 (SD = 31) |
| Test substance (µg) | ||||
| 20.0 | 37 (SD = 1) | 93 (SD = 5) | 19 (SD = 1) | 5 (SD = 1) |
| 80.0 | 57 (SD = 12) | 123 (SD = 8) | 24 (SD = 5) | 8 (SD = 2) |
| 320 | 97 (SD = 10) | 136 (SD = 12) | 31 (SD = 2) | 11 (SD = 2) |
| 1280.0 | 0 (SD = 0) | 151 (SD = 8) | 29 (SD = 4) | 7 (SD = 2) |
| 5120.0 | <0> | <0> | 12 (SD = 2) | <0> |
| With S9 | ||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | |
| Spontaneous reversion | 58 (SD = 1) | 167 (SD = 4) | 15 (SD = 5) | 9 (SD = 2) |
| Control DMSO | 47 (SD = 12) | 127 (SD = 9) | 16 (SD = 3) | 11 (SD = 2) |
| Positive control | 2367 (SD = 90) | 2740 (SD = 47) | 769 (SD = 43) | 283 (SD = 36) |
| Test substance (µg) | ||||
| 20.0 | 41 (SD = 5) | 126 (SD = 11) | 16 (SD = 2) | 13 (SD = 3) |
| 80.0 | 55 (SD = 12) | 137 (SD = 29) | 11 (SD = 1) | 15 (SD = 3) |
| 320 | 51 (SD = 8) | 141 (SD = 2) | 17 (SD = 3) | 15 (SD = 3) |
| 1280.0 | 115 (SD = 11) | 127 (SD = 14) | 14 (SD = 3) | 13 (SD = 3) |
| 5120.0 | <0> | 133 (SD = 9) | 27 (SD = 2) | <0> |
| Without S9 | ||||
| TA 98 | TA 100 | TA 1535 | TA 1537 | |
| Spontaneous reversion | 24 (SD = 5) | 99 (SD = 14) | 21 (SD = 5) | 6 (SD = 2) |
| Control DMSO | 21 (SD = 1) | 97 (SD = 6) | 22 (SD = 4) | 3 (SD = 1) |
| Positive control | 756 (SD = 108) | 2597 (SD = 116) | 2257 (SD = 110) | 291 (SD = 8) |
| Test substance (µg) | ||||
| 20.0 | 24 (SD = 2) | 105 (SD = 9) | 26 (SD = 5) | 7 (SD = 1) |
| 80.0 | 46 (SD = 5) | 109 (SD = 4) | 19 (SD = 4) | 6 (SD = 2) |
| 320 | 77 (SD = 9) | 152 (SD = 15) | 21 (SD = 7) | 14 (SD = 4) |
| 1280.0 | 0 (SD = 0) | 158 (SD = 7) | 16 (SD = 4) | 6 (SD = 2) |
| 5120.0 | <0> | <0> | 13 (SD = 1) | <0> |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
There are no in vitro studies available on Direct Violet 90.
Two Ames tests are available on similar substance 01 one test (Huntsman, 1995) was performed on strain: TA 1535, TA 1537, TA 98, and TA 100, and one test (R&CKFT, 2020) was performed on strain TA98, TA100, TA102, TA1535, TA1537. It was performed also a nitroreductase deficient strains, TA 98NR and TA 100 NR. There are also other Ames studies performed on other similar substances (02 and 04) belongings to the same dye family which showed the same results.
Having the first AMES showing some positivity (Huntsman, 1995), in order to avoid a new study with vertebrate animals, we included in the OECD 422 test (Annex VIII) also a part concerning the mutagenicity potential of the substance, in particular the analysis of the micronuclei. Same animals used for OECD 422 were investigated and no new animals were killed for assessing this endpoint. The examination of the bone marrow and peripheral blood was performed with females only. At the termination of the Dose Range Finding Experiment with the test substance performed for Combined Study, the bone marrow harvesting and the blood taking was done. Evaluation and interpretation of results was performed according to OECD Test Guideline 474 (1997) Mammalian Erythrocyte Micronucleus Test. Statistically significant increase in the number of micronucleated polycbromatic erythrocytes in the bone marrow and normochromatic erythrocytes in peripheral blood compared to the control was not recorded at any dose level. Negative results in the micronucleus test indicate that under the test conditions, the test substance does not produce micronuclei in polychromatic erythrocytes in bone marrow and normochromatic erythrocytes in peripheral blood of rat. The test substance is considered to be non-genotoxic under the condition of the test (REACH&Colours Kft, 2014).
Based on the available information on gene mutation on similar substances and in order to further and completely assess its gene mutation properties in different tissues of the animal, a Comet Assay, OECD 489, on Acid Brown 282 EC 274-490-1 was presented as testing proposal and it will be also used in read across for assessing the in vivo potential gene mutation properties of Acid Violet 90.
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
According to the CLP Regulation (EC 1272/2008), for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:
- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or
- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.
The test substance did not show any reasons of concern in the test performed.
In conclusion, the substance is not classified for genetic toxicity according to the CLP Regulation (EC 1272/2008).
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