2-Naphthalenol, 1-[[4-(phenylazo)phenyl]azo]-, ar-heptyl ar',ar''-Me derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are a total of 8 bacterial mutagenicity assays available. There are variations in the methodologies, strains used and activation systems. There is also a chromosome aberration assay (cultured human lymphocytes)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosome Abberation

Species / strain / cell type:

lymphocytes: Human

Metabolic activation:

with and without

Metabolic activation system:

Rat liver-derived metabolic activating system (S-9 mix)

Test concentrations with justification for top dose:

The concentrations used in the main study, for all exposure regimes, were 8, 80 and 800 ug/ml (the maximum practicable concentration).

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: chlorambucil

Details on test system and experimental conditions:

Culture establishment:
Human peripheral blood was obtained by venepuncture from a healthy, non-smoking, male, human volunteer not currently taking any medication, and collected in heparinised vessels. Small inocula of whole blood (0.5 ml) were added to tubes containing 9.0 ml of culture medium and 0.5 ml phytohaemagglutinin to stimulate lymphocytes to divide. The tubes were sealed and incubated at 37°C with occasional shaking to prevent clumping. After 48 hours of incubation, cultures were treated as described below.

Treatment of cultures:
Each 48-hour culture was centrifuged, the supernatant removed and the cell pellet resuspended in culture medium (9.5 ml, or 8.5 ml for cultures subsequently to contain S-9 mix). Freshly prepared S-9 mix (1.0 ml) was then added to the appropriate cultures and an aliquot (20 ul) of test solution, solvent or positive control solution was added to the relevant cultures. All cultures were incubated at 37°C in a shaking water bath, for three hours. After this initial exposure period, the cultures treated in the absence of S-9 mix were transferred to a 37°C incubator for the remainder of the exposure period. Cultures treated in the presence of S-9 mix and appropriate non-activated cultures were centrifuged and the cells washed twice with Hanks' Balanced Salt Solution (5m1) to remove the test compound and S-9 mix. The cells were then resuspended in culture medium (9.5 ml), and the cultures incubated at 37°C, under static conditions, for a further 21 hours.

Culture harvesting:
Three hours before termination, cell division was arrested by the addition of the spindle poison Colcemid to each culture (to a final concentration of 0.4 ug/ml). The tubes were capped and left to incubate for a further three hours. The cells were then harvested by low speed centrifugation (1400 r.p.m. for 5 minutes) and the pellets of cells thus collected were resuspended in hypotonic potassium chloride solution (0.56%) for ten minutes, centrifuged again and later fixed in freshly prepared methanol : glacial acetic acid fixative (3 : 1 v/v).

Slide preparation:
After two further changes of fixative, the tubes were centrifuged, the supernatant removed and the cell pellet resuspended in a few drops of fresh fixative. Single drops of the cell suspension were transferred to clean, moist, grease-free glass slides, and the slides were left to air-dry. Two or four slides (for the preliminary toxicity test or main cytogenetic test respectively) were made from each culture, stained for ten minutes in Giemsa stain (1 in 10 in Sorensen's buffer, pH 6.8), washed in buffer and left to air-dry. Permanent mounts were made using DPX mountant after clearing in xylene.

Preparation of culture media:
443 ml RPMI 1640 medium with HEPES, sodium bicarbonate, and L-glutamine
50 ml Foetal calf serum
5 ml Heparin (100i.u./m1)
2 ml Penicillin/streptomycin (5000 i.u./ml; 5000 ug/ml)

Preparation of a post-mitochondrial fraction and S-9 liver enzyme mix:
1. Post-mitochondrial fraction
Young male CD rats, c. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent, England. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity. Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KC1, then homogenised with one volume of the same medium in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9000 G for 10 minutes, and supernatants collected and stored at -180°C until required for preparation of the S-9 mix. Supernatant is used within six months of preparation.

2. S-9 mix
0.1M KH2PO4-Na2HPO4 buffer 7.4 ml
0.4M MgC12.6H20/1.65M KC1 aqueous solution 0.2 ml
0.1M NADP, sodium salt, in aqueous solution 0.4 ml
0.1M glucose-6-phosphate, sodium salt, in aqueous solution 0.5 ml
Supernatant from liver homogenate (protein content 102 mg/ml) 1.5 ml

Protein concentration in S-9 mix : 15.3 mg/ml

Evaluation criteria:

Main cytoqenetic test:
- chromosomal analysis and mitotic index. At least two slides from each culture were randomly assigned code numbers by a person not subsequently engaged in the study. Care was taken to ensure that all unique identifications remained concealed to eliminate bias. The slides were examined under a low power (x 10 objective) and those areas judged to be of sufficient technical quality to permit scoring were located and examined under high power (x 100, oil immersion objective). From 100 metaphases (with 46 centromeres) the following characters were recorded:
- chromosome number
- all chromosomes normal or some aberrant
- specific types and numbers of aberrations
Scoring followed the recommendations of the Ad Hoc committee of the Environmental Mutagen Society and the Institute for Medical Research (1972). Morphological observations were scored as follows:
Gap
Break
Fragment
Exchange
Multiple aberrations
Endoreduplication
Pulverised metaphases
Polyploidy

To examine the toxicity of the test compound to dividing lymphocytes, approximately 1000 cells were scored and the mitotic index calculated.
When all examinations had been completed, the coding was broken and the results collated. Frequencies of aberrant metaphases (cells showing one or more aberrations) were calculated for each culture scored, both including and excluding gap-type aberrations.

Statistics:

Statistical analysis:
The Fisher Exact Probability test is a useful nonparametric technique for analysing data when comparing two small independent samples. It is used when the scores for the samples all fall into one or other of two mutually exclusive classes. The test determines whether the two groups differ in the proportions with which they fall into the two classifications. Data from each treatment was compared with the respective solvent control group with or without activation.

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

After 48 hours, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope. This event is believed to be indirectly associated with cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
Precipitation of test compound was apparent in cultures treated with 2-NAPA at 800 ug/ml. When slides were prepared, precipitated compound was apparent in the cell pellet of all cultures treated at 32, 160 and 800 ug/ml. Slides were prepared and stained from all cultures. Inspection of the slide preparations showed no real reduction in mitotic activity (compared to solvent control values) at any 2-NAPA concentration tested, except for a single culture treated at 160 ug/ml in the presence of S-9 mix, which was seen to contain cells but no metaphases. As no such effect was apparent in cultures treated at 800 ug/ml, this was not considered to be a treatment-related effect. Following the findings of the preliminary toxicity test, the 2-NAPA concentrations selected for testing in the main cytogenetic study were 8, 80 and 800 ug/ml for all exposure periods.

Main cytogenetic test:
Precipitation of test compound was apparent in all cultures treated at 800 ug/ml; the supernatant of all cultures treated at 80 ug/ml for 3 hours showed intense red colouration at the end of the exposure period.

Evidence of toxicity:
Initially, mitotic indices were scored for all cultures. No clear depression of mitotic index in test cultures was apparent in either the presence or absence of S-9 mix following any exposure period. However, after 48 hours of exposure, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope (the chromosomes were 'fuzzy'). This event has been seen in other studies performed in this laboratory with other (unrelated) test materials at toxic concentrations, and it is believed to be indirectly associated with cytotoxicity.

Incidence of chromosomal aberrations: comparison of treated and control cultures
One hundred metaphases were scored from each culture. In the absence of S-9 mix, cultures exposed to the solvent (acetone) for three hours only showed incidences of aberrant metaphases of 5 and 6% with a mean of 5.5%. Corresponding values for cultures treated with 2-NAPA were 3 and 4%, mean 3.5% (at 8 ug/ml), 6% in both cultures tested at 80 ug/ml and 5 and 8%, mean 6.5% (at 800 ug/ml). In the presence of S-9 mix, cultures exposed to acetone for three hours only showed incidences of aberrant metaphases of 5 and 6% with a mean of 5.5%. Corresponding values for cultures treated with 2-NAPA were 4% in both cultures treated at 8 ug/ml, 5 and 13%, mean 9.0% (at 80 ug/ml) and 4 and 8%, mean 6.0% (at 800 ug/ml).
Following 24 hours of exposure in the absence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases of 7 and 11% with a mean of 9.0%. Corresponding values for cultures treated with 2-NAPA were 6 and 9%, mean 7.5% (at both 8 and 80 ug/ml) and 9 and 10%, mean 9.5% (at
800 ug/ml). A similar lack of response to 2-NAPA treatment, at the above exposure periods, was apparent when gaps were excluded from consideration. These observations are supported by statistical analysis; no statistically significant increases in aberrant cell frequencies over control values were seen in cultures exposed to 2-NAPA at any concentration for either 3 or 24 hours whether including or excluding gaps (p>0.05 in all cases).
Following 48 hours of exposure in the absence of S-9 mix, solvent control cultures showed incidences of aberrant metaphases of 7 and 8% with a mean of 7.5%. Corresponding values for cultures treated with 2-NAPA were 5 and 7%, mean 6.0% (at 8 ug/ml), 9 and 12%, mean 10.5% (at 80 ug/ml) and
17 and 19%, mean 18.0% (at 800 ug/ml). When gap-type aberrations were excluded, solvent control cultures showed incidences of aberrant metaphases of 0 and 2% with a mean of 1.0%. Corresponding values for cultures treated with 2-NAPA were 0 and 1%, mean 0.5% (at 8 ug/ml), 1% in both cultures treated at 80 ug/ml and 2 and 4%, mean 3.0% (at 800 ug/ml).
No statistically significant increases were seen at any test concentration including gaps, or at 8 and 80 ug/ml excluding gaps (p>0.05). The observed increase in aberrant cell frequency, including gaps, in cultures exposed at 800 ug/ml was seen to be statistically significant (0.01>p>0.001).
When compared to the historical control range for this laboratory (shown below), solvent control cultures harvested after 24 and 48 hours of exposure to acetone showed a small excess of gap-type aberrations. The reason for this is unclear, but in view of the limited magnitude of this excess, the questionable biological significance of gaps and the low frequency of other types of aberration in all solvent control cultures (within the historical control range), this is not considered to impair the validity of the study in any way.

Solvent (acetone) controls: aberrant cell frequencies:
Historical values (600 cells): +S-9 mix 1-6% including gaps, 0-4% excluding gaps
Historical values (600 cells): -S-9 mix 2-6% including gaps, 0-3% excluding gaps

Positive controls:
Treatment with the known clastogen, cyclophosphamide, in the presence of S-9 mix, produced a marked increase in aberrant cell frequency, giving values of aberrant metaphases of 38 and 46%, with a mean of 42.0% including gaps, and values of 18 and 32%, mean 25.0% excluding gaps. Statistical analysis confirmed that this group had significantly more aberrant metaphases than the activated solvent controls whether gap-type aberrations were excluded or not (p < 0.001). Without S-9 mix, cyclophosphamide produced no increases in aberration frequencies compared with non-activated solvent controls whether gaps were included or not (p > 0.05).
Treatment with the known clastogen, chlorambucil, in the absence of S-9 mix showed increased aberrant cell frequencies following all exposure periods as shown below:

Exposure period Including gaps
(hours) Indiv. values p-value
3 16, 23 p<0.001
24 26, 29 p<0.001
48 16, 24 p<0.001

Exposure period Including gaps
(hours) Indiv. values p-value
3 5, 8 0.01>p>0.001
24 13, 14 p<0.001
48 5, 8 0.01>p>0.001

Polyploidy:
The incidence of polyploid cells throughout the study was low, and no treated group showed a real increase compared to concurrent solvent control cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All dose levels tested.
negative without metabolic activation All dose levels tested.

It is concluded that exposure to 2-NAPA at the highest tested concentration induced gap-type aberrations in human lymphocytes. This action was observed only in the absence of S-9 mix after prolonged (48 hours) exposure. In view of the questionable biological significance of gap-type aberrations, 2-NAPA would not be designated a clastogen on the basis of this evidence.

Executive summary:

The effects on chromosomal structure of exposure to 2-Naphthalenol [(phenylazo)phenyl]azo heptyl derivatives (2-NAPA) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activating system (S-9 mix): without S-9 mix cells were exposed for 3, 24 and 48 hours, with S-9 mix exposure was limited to three hours. Treatments were established by the addition of test solutions (in acetone) to 48 hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, Colcemid (to a final concentration of 0.4 ug/ml), three hours before the cells were harvested; slides were then prepared for microscopic analysis.

Mitotic indices were calculated for each culture: these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture, and the frequencies of cells with one or more aberrations were calculated; these aberrant cell frequencies were calculated both including and excluding gap-type aberrations.

Following a preliminary test to assess toxicity of 2-NAPA to dividing lymphocytes,the concentrations used in the main study, for all exposure regimes, were 8, 80 and 800 ug/ml (the maximum practicable concentration).

The main test also incorporated solvent (acetone), and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All control/test concentrations were established in duplicate cultures.

Consideration of mitotic index data showed no direct evidence of toxicity of 2-NAPA to dividing lymphocytes at any tested concentration in either the absence or presence of S-9 mix. However, after 48 hours of exposure, both cultures containing 2-NAPA at 800 ug/ml and one containing 80 ug/ml produced a number of metaphases which could not be clearly visualised under the light microscope (the chromosomes were 'fuzzy'). This event has been seen in other studies performed in this laboratory with other (unrelated) test materials at toxic concentrations, and it is believed to be indirectly associated with cyotoxicity.

No biological or statistically significant increases in the frequency of aberrant cells, over solvent controls, were recorded at any concentration following 3 or 24 hours of exposure (p>0.05); this was true whether gap-type aberrations were included in or excluded from analysis.

Following 48 hours of exposure, an increase in aberrant cell frequency was apparent in cultures treated with 2-NAPA at 800 ug/ml, but only when gaps were included; statistical analysis showed this increase to be significant (0.01>p>0.001)

The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in chromosomal damage over concurrent vehicle controls for all exposure regimes; as expected, cyclophosphamide was active only in the presence of S-9 mix.

It is concluded that exposure to 2-NAPA at the highest tested concentration induced gap-type aberrations in human lymphocytes. This action was observed only in the absence of S-9 mix after prolonged (48 hours) exposure. In view of the questionable

biological significance of gap-type aberrations, 2-NAPA would not be designated a clastogen on the basis of this evidence.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1983

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment. Page 11 is missing from the report which includes information on Historical Control, evaluation criteria and statistics information, therefore the study was assigned a reliability of 3.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. Coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Dose levels for test article 7590B, Lot #83-0176, in the plate assay were 75, 250, 750, 2500 and 7500 ug/plate. There were 0.10 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.
A second assay was conducted to verify results obtained with test article 7590B, Lot #83-0176, with and without metabolic activation in strains TA1538 and TA98 of Salmonella typhimurium. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Asceptic Technique:
All asceptic techniques, where possible, were carried out in the Baker NCB6 Hood.

Storage:
The tester strains were maintained in quadruplicate at a minimum of -60°C, and served as a master and stock culture.

Working Cultures:
Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50 ml of oxoid broth and grown for 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker.

Master Plates:
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were enployed as a source of inoculum for mutagenesis testing. Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of
minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the
histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures were then streaked onto the surface of the plates. The plates were incubated over night at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures. Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar::
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes (13 x 100 mm) with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgCl2; 1.65 M KCl 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 335 - 395 ul
S-9 Fraction 40 - 100 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco 32M). The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies. (Artek Counter Model 880).

Test Article:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Stability and Purity:
Test article 7590B, Lot #83-0176, produced a heavy red precipitate at the 25 and 75 mg/ml levels when added to the aqueous top agar solution. There was no apparent change in the physical state of the control articles during the assay. The purity of the test article was the responsibility of the sponsor.

Evaluation criteria:

No data.

Statistics:

No data.

Species / strain:

other: TA1535, TA1537 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

TA1538 at doses of 75 and 250 ug/plate and TA98 at doses of 75, 250 and 750 ug/plate.

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

TA1538 at doses of 750, 2500 and 7500 ug/plate and TA98 at doses of 2500 and 7500 ug/plate.

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.3, 10, 3.3 and 1 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco Model 32M or Model 4). The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 showed excessive inhibition at the 10,000 ug/plate level.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains at all doses.
negative with metabolic activation TA1535, TA1537 and TA100 at all dose levels.
negative with metabolic activation TA1538 at doses of 75 and 250 ug/plate and TA98 at doses of 75, 250 and 750 ug/plate.
positive with metabolic activation TA1538 at doses of 750, 2500 and 7500 ug/plate and TA98 at doses of 2500 and 7500 ug/plate.

The results for test article 7590B, Lot #83-0176, Automate Red B Standard Non-volatile, were positive in strain TA1538 of Salmonella typhimurium with metabolic activation at levels of 750, 2500 and 7500 ug/plate and in strain TA98 of Salmonella typhimurium with metabolic activation at 2500 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Executive summary:

Test article 7590B, Lot #83-0176 was evaluated for genetic activity in the Ames Salmonella typhimurium Assay. Test article 7590B, Lot #83-0176, was soluble in Dimethylformamide. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 showed excessive inhibition at the 10,000 ug/plate level. Dose levels for test article 7590B, Lot #83-0176, in the plate assay were 75, 250, 750, 2500 and 7500 ug/plate. There were 0.10 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

A second assay was conducted to verify results obtained with test article 7590B, Lot #83-0176, with and without metabolic activation in strains TA1538 and TA98 of Salmonella typhimurium. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

The results for test article 7590B, Lot #83-0176, Automate Red B Standard Non-volatile, were positive in strain TA1538 of Salmonella typhimurium with metabolic activation at levels of 750, 2500 and 7500 ug/plate and in strain TA98 of Salmonella typhimurium with metabolic activation at 2500 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because a positive result was found in in vitro gene mutation study in bacteria

Reference 4

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:

1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. No information on statistics reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Activating system derived from rat liver (S-9 mix)

Test concentrations with justification for top dose:

50, 158, 500, 1580 and 5000 µg/plate

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

Test organisms:
Cultures of the histidine-dependent strains of Salmonella typhimurium were derived from cultures provided by Prof. Bruce Ames, University of California.
Cultures of the tryptophan-dependent strain WP2 uvrA of Escherichia coli were derived from cultures provided by Dr. Michael Green, Cell Mutation Unit, Sussex University.
Cultures of all organisms were prepared by overnight incubation of reshly inoculated nutrient broth (Oxoid No.2).

Checking of tester strains:
New batches of frozen culture stocks, stored at -80°C, are prepared at intervals from a central stock held in a liquid nitrogen refrigerator. Samples from each new batch are thawed at least 24 hours after freezing and checked for appropriate amino acid requirement and characteristic spontaneous reversion rate.

Summary of experimental design:
Plates 1 and 2 check sterility of the S-9 mix and the test material.
Plates 3 to 7 assess mutagenic activity of the test material in the presence of S-9 mix.
Plates 9 to 13 assess mutagenic activity of the test material in the absence of S-9 mix.
Plates 8 and 14 indicate the spontaneous reversion rate together with any effect of solvent with and without activation.
Plates 15 to 17 check the activity of the S-9 mix and the sensitivity of the bacteria to known mutagens.
Plates 18 estimate the total number of viable cells in the bacterial suspension.

Preparation of post-mitochondrial fraction and S-9 liver enzyme mix:
1. Post-mitochondrial fraction:
Young male CD rats, ca. 200 g bodyweight, were obtained from Charles River Breeding Laboratories (U.K.), Margate, Kent. Aroclor 1254 (500 mg/kg bodyweight in corn oil) was administered as a single intraperitoneal injection to induce microsomal enzyme activity.
Four days after treatment, the animals were fasted overnight and then killed by cervical dislocation. The livers were removed, washed in cold 0.15M KCl, then homogenised with more of the same medium (approximately 3 ml per g wet liver) in a Potter-Elvehjem homogeniser. Homogenates were centrifuged at 9000 g for 10 minutes and supernatants collected and stored at -80°C until required for preparation of the S-9 mix. Supernatant is used within 3 months of preparation.

2. S-9 mix Quantity (ml)
0.1M NADP, sodium salt, in aqueous solution 0.4
0.1M glucose-6-phosphate, sodium salt, inaqueous solution 0.5
0.4M MgC12.6H20/1.65M KC1 aqueous solution 0.2
Supernatant from liver homogenate 1.5
0.1M KH2PO4-Na2HPO4 buffer (pH 7.4) to a final volume of 10.0

Preparation of media:
1. Amino-acid-deficient top-agar
Oxoid agar No. 1 0.4 g
NaCl 0.5 g
Distilled water to 100 ml
The medium was autoclaved for 15 minutes at 121°C and 15 lb/in2 pressure. After cooling to 45°C, 10 ml of a sterile aqueous solution of 0.5 mM biotin - 0.5 mM histidine.HCl or 0.5 mM tryptophan was added aseptically.

2. Amino-acid-rich medium
Oxoid agar No. 1 10 g
Oxoid nutrient broth No. 2 25 g
Distilled water to 1 litre
The medium was autoclaved for 15 minutes at 121°C and 15 lb/in squared pressure.

3. Minimal medium
This was Vogel-Bonner minimal "E" medium with 2% dextrose, prepared as follows:
Solution A (VBE Salts):
MgSO4.7H20 1 g
Citric acid 10 g
K2HPO4 50 g
NaNH4PO4.4H20 17.5 g
Distilled water to 100 ml
The solution was autoclaved for 15 minutes at 121°C and 15 lb/in squared pressure.

Solution B (20% dextrose):
Dextrose 40 g
Distilled water to 200 ml
The solution was autoclaved for 10 minutes at 121°C and 15 lb/in squared pressure.

Solution C (Agar base):
Oxoid agar No. 1 5g
Distilled water to 440 ml
Solution C was autoclaved for 15 minutes at 121°C and 15 lb/in squared pressure. After cooling to 55°C, 10 ml Solution A and 50 ml solution B were added aseptically.

Pour-plate assay for mutagenesis:
An aliquot (0.1 ml) of each concentration of 2-NAPA was placed in a sterile tube. Molten, amino-acid-deficient top-agar (2 ml) and bacterial suspens on (0.1 ml), maintained at 45°C, was then added. The tubes were mixed by inversion and 0.5 ml rat liver microsomal preparation (S-9 mix) was added where appropriate. The tubes were again inverted to mix thoroughly and the contents poured onto plates containing solidified minimal medium (20 ml).
Further plates were prepared without the inclusion of the test organisms to verify the sterility of the S-9 mix and the test material. A control series of plates was prepared to confirm the inability of acetone (0.1 ml) to induce reversion in the bacterial strains, and to provide a measure of the spontaneous mutation rates.
Aliquots (0.1 ml) of a 10E6 dilution of culture were spread on the surface of plates of complete medium to measure the viability and cell-density of each culture.

Evaluation criteria:

All plates were prepared in triplicate, allowed to solidify and incubated at 37°C for 2 days. After incubation, numbers of revertant colonies were counted, either manually or with a Biotran II automatic colony counter. Total colonies on nutrient plates were counted in the same way. Growth of the background lawn of non-revertant cells on minimal plates was verified.
Each test, in each strain, was conducted on two separate occasions.
All plates and tubes were identified by the use of numbers indelibly marked on the plates and test tube racks.

Statistics:

No data.

Species / strain:

other: S. typhimurium T98, T100 and T1537

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

5000 ug per plate

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

50, 158, 500 and 1580 ug per plate

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

other: Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary toxicity test:
A solution of 2-NAPA was prepared in acetone at 25 mg/ml, and an aliquot of this solution (0.2 ml) was transferred to a sterile tube containing molten, amino-acid-deficient top-agar (2.0 ml) maintained at 45°C. An additional aliquot (0.1 ml) of the test material solution was similarly transferred to another tube of molten top-agar (2.0 ml).
Three serial ten-fold dilutions in molten top-agar were prepared from each of these preparations, giving a series of eight different concentrations of test material from 2.5 ug to 5 mg per plate. All tubes were inoculated with an overnight culture of strain TA 98 (0.1 ml) and overlaid onto minimal medium plates. Control plates were prepared containing top-agar and culture alone, top-agar, acetone (0.2 ml) and culture, and top-agar and test material
(0.1 ml) without bacterial culture.
The procedure was repeated using strain WP2 uvrA.
The plates were incubated at 37°C for 2 days and were then examined for the presence of a background lawn of non-revertant colonies; toxicity of the test material is shown by absence or thinning of the background lawn. The level of test material chosen as the top level for pour-plate tests is normally the lowest level causing visible thinning of the lawn. (In the absence of such thinning, a top level of 5 mg/plate was selected).
The control plates were checked for the absence of growth on sterility checks or normal counts on negative controls in the presence and absence of the vehicle.
No visible thinning of the background lawn of non-revertant cells was obtained following exposure to 2-NAPA. A top exposure level of 5 mg per plate was therefore selected for use in the main assays.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
ambiguous with metabolic activation Positive in T98, T100 and T1537. Negative in T1535 and E.coli WP2 uvrA.
ambiguous without metabolic activation Positive in T98. Negative in all other strains tested. E coli also negative.

The test material, 2-NAPA, exhibited mutagenic activity in this study, inducing frameshift mutations following metabolic activation (and weak activity without metabolic activation).

Executive summary:

2-NAPA was examined for mutagenic activity in four histidine-dependent auxotrophs of Salmonella typhimurium, strains TA 98, TA 100, TA 1535 and TA 1537, and one tryptophan-dependent auxotroph of Escherichia coli, strain WP2 uvrA, using pour-plate assays. The procedures used complied with OECD Guidelines for Testing of Chemicals Nos. 471 and 472 (issued 1983), EPA Toxic

Substances Control Act Test Guideline § 798.5265 (first issued 1985, amended 1987) and the Revised Chemical Substances Control

Law (1987) of the Japanese Ministry of Health and Welfare and Ministry of International Trade and Industry. Each test, in each strain, was conducted on two separate occasions.

The studies, which were conducted in the absence and presence of an activating system derived from rat liver (S-9 mix), employed a

range of levels of 2-NAPA from 50 to 5000 ug per plate, selected following a preliminary toxicity test in strains TA 98 and WP2 uvrA, and included solvent (acetone) controls with and without S-9 mix.

Dose-related increases in reversion to prototrophy were obtained in strains TA 98, TA 100 and TA 1537 following exposure to 2-NAPA in the presence of S-9 mix. Small increases were also seen in strain TA 98 without S-9 mix.

Increases in revertant colony numbers over control counts reached 18.4- and 21.4-fold (TA 98) and 4.5- and 6.5-fold (TA 1537) in

tests 1 and 2 respectively, and 2.9-fold (TA 100, both tests) in the presence of S-9 mix. In the absence of S-9 mix, the increases

were 2.7- and 2.4-fold (TA 98) in tests 1 and 2 respectively.

Marked increases in the number of revertant colonies were induced by the known mutagens benzo[a]pyrene, 2-nitrofluorene, 2-aminoanthracene, 9-aminoacridine, ENNG and sodium azide when examined under similar conditions.

It was concluded that 2-NAPA exhibited mutagenic activity in this study, inducing frameshift mutations following metabolic activation (and weak activity without metabolic activation).

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. Coli not tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Dose levels for test article 7590A, Lot #83-0175 in the plate assay were 75, 250, 750, 2500 and 7500 ug/plate. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

A second study was conducted to verify results obtained for test article 7590A, Lot #83-0175 in strains TA1538 and TA98 with metabolic activation. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Asceptic Technique:
All asceptic techniques, where possible, were carried out in the Baker NCB6 Hood.

Storage:
The tester strains were maintained in quadruplicate at a minimum of -60°C, and served as a master and stock culture.

Working Cultures:
Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50 ml of oxoid broth and grown for 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker.

Master Plates:
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing. Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of
minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the
histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures were then streaked onto the surface of the plates. The plates were incubated over night at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures. Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes (13 x 100 mm) with kaputs were labeled and placed into a Fisher IsotempR Dry Bath (No. 145) at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liyer homogenate obtained from Aroclor 1254- treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgCl2; 1.65 M KCl 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 335 - 395 ul
S-9 Fraction 40 - 100 ul

The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific ThelcoR 32M). The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies. (Artek Counter Model 880).

Test Article:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Stability and Purity:
Test article 7590A, Lot #83-0175 produced a heavy red precipitate when added to an aqueous top agar solution at the 25 and 75 mg/ml level. There was no apparent change in the physical state of the control articles during the assay. The purity of the test article was the responsibility of the sponsor.

Evaluation criteria:

Data Reporting:
Standard form (Data Summary Sheet). In scoring the assay, the positive and negative controls were first evaluated. If the control values did not fall within the acceptable historical values, the remaining plates were not scored and the assay was repeated.

Evaluation Criteria:
In most tests with the Salmonella/microsome assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical will be considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical will be considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

TA1538 at dose of 75 ug/plate and TA98 at doses of 75 and 250 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

TA1538 at doses of 250, 750, 2500 and 7500 ug/plate and TA98 at doses of 750, 2500 and 7500 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study up to 3333 ug/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.3, 10, 3.3 and 1 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator (GCA/Precision Scientific Thelco Model 32M or Model 4). The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 showed excessive inhibition at the 10,000 ug/plate level.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative without metabolic activation All strains at all doses.
negative with metabolic activation TA1535, TA1537 and TA100 at all dose levels.
negative with metabolic activation TA1538 at 75 ug/plate and TA98 at 75 and 250 ug/plate
positive with metabolic activation TA1538 at 250, 750, 2500 and 7500 ug/plate and TA98 at 750, 2500 and 7500 ug/plate

The results for test article 7590A, Automate Red B Primary, Non-Volatile, Lot #83-0175 were positive in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at 250, 750, 2500 and 7500 ug/plate and in strain TA98 of Salmonella typhimurium with metabolic activation at 750, 2500 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Executive summary:

Test article Automate Red B Primary, Non-Volatile Lot #83-0175 was evaluated in the Ames Salmonella/Microsome Plate Test. Test article 7590A, Lot #83-0175 was soluble in Dimethylformamide. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 showed excessive inhibition at the 10,000 ug/plate level.

Dose levels for test article 7590A, Lot #83-0175 in the plate assay were 75, 250, 750, 2500 and 7500 ug/plate. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

A second study was conducted to verify results obtained for test article 7590A, Lot #83-0175 in strains TA1538 and TA98 with metabolic activation. There were 0.1 ml of S-9 supernatant (42 mg protein per ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

The results for test article 7590A, Automate Red B Primary, Non-Volatile, Lot #83-0175 were positive in strain TA1538 of Salmonella typhimurium with metabolic activation preparation at 250, 750, 2500 and 7500 ug/plate and in strain TA98 of Salmonella typhimurium with metabolic activation at 750, 2500 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment. OECD guideline followed was not specified.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. Coli was not tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Test article Automate Red B was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without
metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.08 ml of S-9 supernatant (32.3 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver metabolic activation preparation. Test article Automate Red B, Lot #1658-77, was re-evaluated in strain
TA100 of Salmonella typhimurium with metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate due to an increased mutation frequency observed at 50 and 166.6 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of Salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for approximately 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 16 hour growth period, 1 ml aliquots of the culture was dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing.
Fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 50 m of Oxoid Nutrient Broth #2 and grown for approximately 16 hours (1-2 x 10E9 cells/ml) at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker . After the 16 hour incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer. Historical data has shown that optical densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked concurrently for the presence of the appropriate genetic markers at the time of the assay.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgC12; 1.65 M KCI 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 355 ul
S-9 Fraction 80 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with the assay. These contamination checks included the top level of test substance, solvent, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Test article Automate Red B produced a precipitate when added to the aqueous top agar solution at the 166.6, 500, 1666.6 and 5000 ug/plate levels. There was no apparent change in the physical state of the control articles during the assay.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the negative control values did not fall within the acceptable historical mean values, the remaining plates were not scored and the assay was repeated.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control
values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 1, 3.3, 10 and 33.3 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results of test article Automate Red B were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable limits of mean historical limits.

Executive summary:

Test article, Automate Red B, Lot #1658-77, was received as a dark red solid and was soluble in DMF. Dose levels in a Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Strains TA1538 and TA100 of Salmonella typhimurium showed no inhibition of bacterial growth at the levels tested.

Test article Automate Red B was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. There were 0.08 ml of S-9 supernatant (32.3 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver metabolic activation preparation. Test article Automate Red B, Lot #1658-77, was re-evaluated in strain TA100 of Salmonella typhimurium with metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate due to an increased mutation frequency observed at 50 and 166.6 ug/plate.

The results for test article Automate Red B were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166.6, 500, 1666.6 and 5000 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of mean historical data.

Reference 7

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Concentrations for test article 7633A, Lot #83-0301, were 100, 333, 1000, 3333 and 10,000 ug/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of Salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
The tester strains were maintained in quaduplicate at a minimum of -60°C, and served as a master and stock culture. In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing. Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures and were then streaked onto the surface of the plates. The plates were incubated overnight at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures.
Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50 ml of oxoid broth and grown overnight at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from
Aroclor 1254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgCl2; 1.65 M KCl 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 335 ul
S-9 Fraction 100 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Test article 7633A, Lot #83-0301, produced a reddish orange precipitate when added to the aqueous top agar solution at the 100, 75, 33.3, 50, 25 and 10 mg/ml levels.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the control values did not fall within the acceptable historical values,
the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

Doses of 2500, 5000 and 7500 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.33, 10, 3.33 and 1.0 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results for test article 7633A, Lot #83-0301, Automate Red B - Simple Amine Dyes, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strain TA1538 of Salmonella typhimurium with metabolic activation at 2500, 5000 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Executive summary:

Test article 7633A, Lot #83-0301, Automate Red B-Simple Amine Dyes, was soluble in DMF at 100 mg/ml. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested. Test article 7633A, Lot #83-0301, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation in the plate assay at doses of 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, due to aberrant values obtained in the assay, additional dose levels of 2500, 3333, 5000, 7500 and 10,000 ug/plate were retested in strain TA1538 with metabolic activation. There were 0.10 ml of S-9 supernatant (42 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

The results for test article 7633A, Lot #83-0301, Automate Red B - Simple Amine Dyes, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strain TA1538 of Salmonella typhimurium with metabolic activation

at 2500, 5000 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Reference 8

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

E. coli not tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 fraction

Test concentrations with justification for top dose:

Test article 7633C, Lot #83-0303, was evaluated in tester strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation in the plate assay at doses of 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, due to the aberrant values observed in the assay, additional levels of 1000, 3333, 5000, 7500 and 10,000 ug/plate were evaluated in strains TA1538 and TA98 with metabolic activation preparation. There were 0.10 ml of S-9 supernatant (42 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of
salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
The tester strains were maintained in quaduplicate at a minimum of -60°C, and served as a master and stock culture.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, master plates were employed as a source of inoculum for mutagenesis testing. Master Plates were prepared by spreading 0.1 ml of 0.1 M histidine and 0.5 mM biotin on the surface of minimal glucose agar plates using a sterile glass spreader. For the R-factor strains, 0.1 ml of ampicillin (8 mg/ml) was spread in addition to the histidine and biotin as pressure for retaining the plasmid. The plates were prepared from frozen stock cultures and were then streaked onto the surface of the plates. The plates were incubated overnight at 37°C and were then refrigerated. These plates can be used as a source of inoculum for mutagenesis testing for 2-3 months. New Master Plates were always made from frozen stock cultures.
Fresh cultures for mutagenesis testing were prepared by inoculating a loop of inoculum from Master Plates into 50 ml of oxoid broth and grown overnight at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Tester strains were routinely checked for the presence of the appropriate genetic markers.

Top Agar:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine and 0.5 mM Biotin at a volume of 0.1 ml per ml of agar, and maintained at 45°C until used. Sterile tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All negative and positive control tubes and plates were done in triplicate. All compound-treated tubes and plates were done in duplicate. Using sterile
technique, the following were added to each tube in the following order: 2 ml aliquots of top agar solution, 0.1 ml of tester strain, and 0.1 ml of the
appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden.

Metabolic Activation System:
Tubes requiring metabolic activation have, in addition to the preceding top agar ingredients, an S-9 fraction of rat liver homogenate obtained from
Aroclor l254-treated Sprague Dawley rats. The activation system (S-9 mix) contained the following per ml:
0.4 M MgC12; 1.65 M KCl 20 ul
1 M Glucose - 6 - Phosphate 5 ul
0.1 M NADP 40 ul
0.2 M Phosphate buffer pH 7.4 500 ul
Sterile distilled H2O 335 ul
S-9 Fraction 100 ul
The S-9 fraction was thawed on the day of use and kept cold on ice. 0.5 ml of the S-9 mix was added to the tubes which were then vortexed and poured onto minimal glucose plates. The plates were allowed to harden for several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 - 72 hours, checked for uniform background lawn, and scored by counting revertant colonies on an electronic colony counter interfaced with a computer.

Test Article Purity:
The identity, purity, quality, and strength of the test article is the responsibility of the sponsor.

Test Article Stability:
Test article 7633C, Lot #83-0303, Automate Red B, Purified produced a white precipitate when added to the aqueous top agar solution at the 100, 75, 50 and 33.33 mg/ml levels.

Evaluation criteria:

Data Reporting:
In scoring the assay, the positive and negative controls were first evaluated. If the control values did not fall within the acceptable historical values,
the remaining plates were not scored and the assay was repeated. A summary of the data are presented in the summary data sheet contained in this report.

Evaluation Criteria:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as a reproducible, dose-related increase in the number of histidine-independent colonies. This dose-response relationship occasionally necessitates slight modification of the original doses in a repeat assay. If the solvent control is within one standard deviation of the historical mean (See Historical Data) for control values and the test chemical produces the highest increase equal to or greater than three times the solvent control value, the test chemical is considered mutagenic. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

other: TA1538 and TA98

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

5000 and 7500 ug/plate

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Ames Assay used two of the histidine auxotrophs of Salmonella typhimurium TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains. The test compound was prepared to a concentration of 100 mg/ml. Logarithmic dilutions of this stock solution were made in the appropriate solvent to give the following concentrations: 33.33, 10, 3.33 and 1.0 mg/ml. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 45°C. All control and treated tubes and plates were done in duplicate. Using sterile technique, the following were added to each tube: 2 ml aliquots of top agar solution, 0.1 ml of tester strain and 0.1 ml of the appropriate concentration of the test compound. The tubes were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested. A red precipitate was observed in the 3333 and 10,000 ug/plate dose levels.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results for test article 7633C, Lot #83-0303, Automate Red B, Purified, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strains TA1538 and TA98 of Salmonella typhimurium with metabolic activation at 5000 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Executive summary:

Test article 7633C, Lot #83-0303, Automate Red B Purified, was soluble in DMF at 100 mg/ml. Dose levels in the Preliminary Toxicity Screen were 100, 333, 1000, 3333 and 10,000 ug/plate. Tester strains TA1538 and TA100 showed no inhibition at all levels tested. Test article 7633C, Lot #83-0303, was evaluated in tester strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without metabolic activation preparation in the plate assay at doses of 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, due to the aberrant values observed in the assay, additional levels of 1000, 3333, 5000, 7500 and 10,000 ug/plate were evaluated in strains TA1538 and TA98 with metabolic activation preparation. There were 0.10 ml of S-9 supernatant (42 mg protein/ml) per 1.0 ml of S-9 mix used in the rat liver microsomal activation system.

The results for test article 7633C, Lot #83-0303, Automate Red B, Purified, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at 100, 333, 1000, 3333 and 10,000 ug/plate. In addition, the results were negative in strains TA1538 and TA98 of Salmonella typhimurium with metabolic activation at 5000 and 7500 ug/plate. All solvent and positive controls used in the evaluation of the test article were within the acceptable range of historical data.

Reference 9

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: OECD guideline study performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. E. Coli was not tested in the experiment.

Qualifier:

according to guideline

Guideline:

other: OECD ISBN 92-64-12221-4

Deviations:

no

Remarks:

No deviations from this guideline listed, however, E coli was not tested.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

an uninduced S-9 fraction of hamster liver homogenate

Test concentrations with justification for top dose:

Test article, Automate Red B, Lot #1658-77, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without hamster liver metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate using the methods of the Prival Modification. Strain TA100 was re-evaluated both with and without hamster liver metabolic activation preparation at the same dose levels due to negative control values which did not fall within the limits of acceptable data. There were 0.30 ml of S-9 supernatant (28.25 mg protein per ml) per 1.0 ml of S-9 mix in the hamster liver metabolic activation preparation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMF

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMF

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Anthramine

Details on test system and experimental conditions:

TEST ARTICLE:
The test article, Automate Red B, Lot #1658-77, was received by Pharmakon Research on September 7, 1983 in a metal container and the physical description of the test article upon receipt was described as a dark red solid. Normal precautions were used in handling the test article. Stability and purity of the test article were the responsibility of the sponsor and information as to the stability, purity, expiration date and other technical aspects of the test article was recorded in the sponsor's file, if provided. For the purposes of this study, the test article was stored at room temperature in the container received from the sponsor. All required dilutions were made with dimethylformamide (DMF), Lot #KTJZ, supplied by Mallinckrodt, Inc., Paris, Kentucky, 40361. Dilutions were prepared the day of the test. At the time of testing the test article was described as a dark red solid. There was no apparent change in the physical state of the test or control articles during the assay. Details of the test article preparation are contained in the experimental data section of this report. Dosing solutions were used within four hours of preparation.

Rationale for Test System:
Chemicals capable of inducing mutations have been shown to increase the reversion frequency at the histidine locus in selected tester strains of Salmonella typhimurium with and without the addition of a metabolic activation system.

Test organism storage and maintenance:
Frozen working stock cultures were prepared by scraping a wooden applicator stick over the surface of frozen Master cultures and inoculating the scrapings into 50 ml of Oxoid Broth #2 and grown for approximately 16 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker. Following the 16 hour growth period, 1 ml aliquots of the culture were dispensed into Nunc vials marked with the particular strain and quick frozen in an ethanol-dry ice bath before being stored at a minimum of -60°C.
In order to avoid the effect of surface thawing and re-freezing of frozen permanent vials of bacterial stock, frozen working stock cultures are employed as a source of inoculum for mutagenesis testing.
Fresh cultures for mutagenesis testing were prepared by quick thawing a vial of frozen working stock cultures of each tester strain and transferring the culture to 25 ml of Oxoid Nutrient Broth #2 and grown for approximately 10 hours at 37°C in a New Brunswick Scientific Model G24 Environmental Incubator Shaker . After the 10 hour incubation, samples of culture suspensions were diluted 1:4 in distilled water and optical densities were observed at 650 nm using a Beckman Model 35 Spectrophotometer. Historical data has shown that optical densities of 0.400 or greater are representative of cells in late exponential or early stationary phase of growth. Tester strains were checked for the presence of the appropriate genetic markers on a monthly basis.

Pre-incubation Treatment:
The Prival modified pre-incubation mutation assay used the five histidine auxotrophs of Salmonella typhimurium TA1535, TA1537, TA1538, TA98 and TA100. Sterile tubes (13 x 100 mm) with kaputs were labeled and placed into a Fisher Isotemp Dry Bath (No. 145) at 30°C for pre-incubation treatment. All negative and positive controls and all compound treated tubes and plates were done in triplicate. Using sterile technique, the following were added to each tube in the following order: 0.5 ml of 0.2 M phosphate buffer pH. 7.4 or 0.5 ml of the hamster liver S-9 mix was added per tube, 0.1 ml of tester strain, and 0.1 ml of the appropriate concentration of the test substance or control vehicle solvent. The tubes were incubated at 30°C for 30 minutes without shaking.

Metabolic Activation System:
For metabolic activation, an uninduced S-9 fraction of hamster liver homogenate obtained from male Syrian Golden hamsters prepared according to the method of Ames et al was employed. The activation system (S-9 mix) contained the following per ml: The volume of the S-9 fraction was 300 ul/ml (150 ul/plate).
0.4M MgCl2; 1.65M KCl 20 ul
1M Glucose - 6 - Phosphate 20 ul
280 units G-6-P04 Dehydrogenase 10 ul
0.2M NADP 20 ul
0.2M NADH 10 ul
0.2M FMN* 10 ul
0.2M Phosphate buffer pH 7.4 500 ul
Sterile Distilled H2O 110 ul
S-9 Fraction 300 ul
*FMN was added to the S-9 mix last to prevent the mix from becoming discolored (Personal Communication M.J. Prival). The S-9 fraction is thawed on the day of use and maintained on ice.

Plating Procedure:
Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5 mM histidine - 0.5 mM biotin at a volume of 0.1 ml per
ml of agar, and maintained at 45°C until used. Following pre-incubation, addition of top agar and plate pouring, the plates were allowed to harden for
several minutes. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C (± 2°C) incubator. The plates were incubated for 48 to 72 hours, checked for uniform background lawn, and scored by counting revertant colonies with an Artek Counter (Model 880).

Bacterial Contaminant Control:
To insure the quality of aseptic technique during the assay and also the sterility of solvents, compounds and equipment, standard contamination checks were conducted with each assay. These contamination checks include the top level of test substance, top agar and S-9 mix at the same volumes as in the assay. The test substance or S-9 mix were added to 2 ml of molten top agar supplemented with 0.5mM histidine - 0.5mM biotin and poured onto minimal glucose plates. Top agar alone was also plated on minimal glucose plates. All plating was done in triplicate. Plates were incubated for 48 hours at 37°C (± 2°C) and then scored for contamination.

Test Article Purity:
The identity, purity, quality, and strength of the test article are the responsibility of the sponsor.

Evaluation criteria:

Scoring:
In scoring the assay, the positive and negative controls were first evaluated. If the negative or positive control values did not yield acceptable values, the remaining plates were not scored and the assay is repeated.

Confirmatory Assay:
All equivocal or positive results are repeated in those strains which result in a minimum of a doubling of the solvent control mutation frequency. At the discretion of the sponsor, at additional cost, a confirmatory assay will be conducted for negative results as well.

Evaluation:
In most tests with the Salmonella/Microsome Assay, results are either clearly positive or clearly negative. A positive result is defined as reproducible, dose-related increase in the number of histidine-independent colonies. As an alternative criteria, if the test substance produces the highest increase equal to or greater than three times the solvent control value the test substance will be considered positive. A negative result is defined as the
absence of a reproducible increase in the number of histidine-independent colonies.

Statistics:

A positive result is defined as reproducible, dose-related increase in the number of histidine-independent colonies. As an alternative criteria, if the test substance produces the highest increase equal to or greater than three times the solvent control value the test substance will be considered positive. A negative result is defined as the absence of a reproducible increase in the number of histidine-independent colonies.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study with the exception of TA100 which showed a reduced number of revertants at the 5000 ug/plate level without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels

Cytotoxicity / choice of top concentrations:

not specified

Remarks:

Normal growth observed in preliminary study with the exception of TA100 which showed a reduced number of revertants at the 5000 ug/plate level without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Screen:
The preliminary toxicity screen for the Prival Modified Ames Assay performed with and without metabolic activation used two of the histidine auxotrophs of Salmonella typhimurium, TA1538 and TA100. The preliminary toxicity screen was designed to determine at which levels the compound exhibits toxic effects to the Salmonella typhimurium tester strains both with and without hamster liver metabolic activation preparation. The test compound was prepared to a concentration of 50 mg/ml and five different levels tested for toxicity. Sterile glass tubes with kaputs were labeled and placed into a Fisher Isotemp Dry Bath at 30°C. All control and treated tubes and plates were done in duplicate. Using sterile technique the following were added to each tube: 0.1 ml of tester strain, 0.5 ml of 0.2M phosphate buffer pH 7.4 or 0.5 ml of hamster liver metabolic activation preparation and 0.1 ml of the appropriate concentration of the test compound. The tubes were incubated without shaking at 30°C for 30 minutes. Top agar, used as an overlay, was reconstituted into a molten state and supplemented with 0.5mM histidine - 0.5mM biotin at a volume of 0.1 ml/ml of agar, and maintained at 45°C until used. Following preincubation treatment 2 ml aliquots of molten top agar solution were added to the tubes and they were vortexed and poured onto minimal glucose plates. The sample was evenly distributed on the plate, and the top agar overlay was allowed to harden. The same procedure was repeated for each tester strain. Within an hour the plates were inverted and placed in a dark 37°C incubator. The plates were incubated for 48 hours following which the background lawn and spontaneous revertants were observed and scored as normal growth, inhibited growth or no growth. Inhibition was scored by the presence of pindot colonies and the absence of a confluent lawn of bacteria. The Preliminary Toxicity Screen was executed both with and without hamster liver metabolic activation preparation at dose levels of 50, 166, 500, 1666 and 5000 ug/plate. Strain TA1538 and TA100 of Salmonella typhimurium showed no inhibition of bacterial lawn growth with and without metabolic activation at all the levels tested with the exception of TA100 which showed a reduced number of revertants at the 5000 ug/plate level without metabolic activation. Based on these findings, the highest dose selected for the Plate Incorporation Mutation Assay was 5000 ug/plate.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation All strains at all doses.
negative without metabolic activation All strains at all doses.

The results for test article, Automate Red B, Lot #1658-77, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate.

Executive summary:

Test article, Automate Red B, Lot #1658-77, was received as a dark red solid and was determined to be soluble in dimethylformamide (DMF). A Preliminary Toxicity Screen was executed both with and without hamster liver metabolic activation preparation at dose levels of 50, 166, 500, 1666 and 5000 ug/plate. Strain TA1538 and TA100 of Salmonella typhimurium showed no inhibition of bacterial lawn growth at the levels tested with the exception of TA100 which showed a reduced number of revertants at the 5000 ug/plate level without metabolic activation.. Based on these findings, the highest dose selected for the Plate Incorporation Mutation Assay was 5000 ug/plate.

Test article, Automate Red B, Lot #1658-77, was evaluated in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium with and without hamster liver metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate using the methods of the Prival Modification. Strain TA100 was re-evaluated both with and without hamster liver metabolic activation preparation at the same dose levels due to negative control values which did not fall within the limits of acceptable data. There were 0.30 ml of S-9 supernatant (28.25 mg protein per ml) per 1.0 ml of S-9 mix in the hamster liver metabolic activation preparation.

The results for test article, Automate Red B, Lot #1658-77, were negative in strains TA1535, TA1537, TA1538, TA98 and TA100 of Salmonella typhimurium both with and without metabolic activation preparation at doses of 50, 166, 500, 1666 and 5000 ug/plate. All negative and positive controls used in the evaluation of the test article were within the acceptable range established by this laboratory.

Reference 10

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1999

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. TA 1538 and E. Coli was not tested in the experiment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

The assays were performed by using the preincubation modification of the standard plate incorporation procedure with S. typhimurium strains TA1535, TA1537, TA98, and TA100 in both the presence and absence of a reductive metabolic activation system (containing flavin mononucleotide as the reducing agent and hamster-liver homogenate).

GLP compliance:

yes

Type of assay:

other: Salmonella/microsome preincubation assay under reductive conditions

Target gene:

Histidine loci

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Metabolic activation system:

Uninduced hamster liver homogenate preparation (S-9); 29.3 mg protein/mL

Test concentrations with justification for top dose:

The test article was determined to be soluble at 100 mg/mL in acetone. An initial range-finding experiment was conducted with Salmonella strain TA98 in the presence and absence of a reductive metabolic activation system to determine a suitable dose range for the first mutagenicity experiment. Doses were achieved by preparing a stock solution at 100 mg/mL (5000 μg/plate) and serially diluting it to obtain doses of 1000, 500, 100, 50, and 10 μg/plate. The range-finding experiment was repeated under the same conditions used in the initial range-finder; however, doses of 10, 50, 100, 250, 500, 1000, and 2500 μg/plate were used. For the first mutagenicity experiment, the test article was evaluated using four tester strains, with three plates per dose level, at dose levels of 0.5, 1, 5, 10, 50, and 100 μg/plate in the presence and absence of a reductive metabolic activation system containing 30% hamster liver S-9. The second mutagenicity experiment was conducted at dose levels of 0.5, 1, 5, 10, 50, 100 and 500 ug/plate in the presence and absence of a reductive metabolic activation system.

Vehicle / solvent:

Solvent:
Name: Acetone
Supplier: Mallinckrodt, Paris, KY
CAS No.: 67-68-5
Lot No.: 2440 KTEX
Physical Description: Colorless liquid
Storage Conditions: Room temperature
Stability: 5 years
Purity: 99.6%
Characterization of Solvent: Characterization of the solvent was obtained from the manufacturer's label.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Concurrent solvent controls were performed with each experiment.

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

sodium azide

congo red

other: 9-Aminoacridine hydrochloride, 2-Anthramine, β - Naphthylamine

Details on test system and experimental conditions:

Experimental Design:
The test article was determined to be soluble at 100 mg/mL in acetone. An initial range-finding experiment was conducted with Salmonella strain TA98 in the presence and absence of a reductive metabolic activation system to determine a suitable dose range for the first mutagenicity experiment. Doses were achieved by preparing a stock solution at 100 mg/mL (5000 μg/plate) and serially diluting it to obtain doses of 1000, 500, 100, 50, and 10 μg/plate. The range-finding experiment was repeated under the same conditions used in the initial range-finder; however, doses of 10, 50, 100, 250, 500, 1000, and 2500 μg/plate were used. For the first mutagenicity experiment, the test article was evaluated using four tester strains, with three plates per dose level, at dose levels of 0.5, 1, 5, 10, 50, and 100 μg/plate in the presence and absence of a reductive metabolic activation system containing 30% hamster liver S-9. The second mutagenicity experiment was conducted at dose levels of 0.5, 1, 5, 10, 50, 100 and 500 ug/plate in the presence and absence of a reductive metabolic activation system.

Justification of Dose Levels Selected:
The highest soluble dose level used in the range-finding experiment was 5000 μg/plate. Dose selection for the mutagenicity experiments was made to demonstrate a dose response.

Test System:
The Salmonella/microsome assay is capable of rapidly detecting the mutagenic activity of many materials, including a wide range of chemical classes. Many chemicals that elicit a mutagenic response in the Salmonella assay have been shown to be potentially mutagenic and carcinogenic to humans and laboratory animals. Because the Salmonella assay is a short-term, sensitive, and reliable test for assessing mutagenic potential, its use for genotoxic evaluation of chemicals is appropriate.

The use of reducing agents has been recommended for testing certain classes of chemicals (e.g., azo and diazo compounds) in the Salmonella/microsome assay. Unless reduced, such compounds usually do not elicit mutagenic activity in the standard Ames test. Several reducing agents have been used for testing azo dyes in the Salmonella assay; however, flavin mononucleotide (FMN) (Prival and Mitchell, 1982) is the reducing agent of choice because it is more water soluble and does not interfere with the mammalian metabolic activation system.

In the preincubation procedure, the test chemical is allowed to incubate at physiological temperature with the test organisms in the presence of a metabolic activation system before dilution with the bacterial growth medium. This initial step increases the likelihood that active metabolites that are short-lived or unstable or that occur in low concentrations will react with the test organism's genetic material (Yahagi et al., 1975).

The test method used is that recommended by Prival and Mitchell (1982), which uses a modified metabolic activation system. It differs from the standard plate incorporation assay in 5 ways: (1) uninduced hamster liver S-9 is used instead of the Aroclor 1254-induced rat-liver S-9; (2) the S-9 concentration is increased to 150 μL per plate instead of the recommended maximum of 50 uL per plate; (3) FMN is included in the cofactor mix; (4) the cofactor mix includes exogenous glucose-6- phosphate dehydrogenase, NADH, and 4 times the amount of glucose-6-phosphate; and (5) the top agar is added to the test tube after a 30-minute preincubation period at 30°C.

Indicator Organisms:
Species: Salmonella typhimurium LT2
Strains: TA1535, TA1537, TA98, and TA100
Source: Dr. Bruce Ames, University of California, Berkeley

Test System Identification:
The strains were analyzed for their genetic markers and for the presence of the plasmid whenever experiments were performed.

Culture Conditions:
The indicator strains were kept frozen at -80°C in nutrient broth supplemented with 10% sterile glycerol. New frozen stock cultures were made from single colony isolates. Cultures were inoculated into 50 mL Oxoid Nutrient Broth No. 2 (CM 67) and allowed to sit unshaken for 2 to 4 hr, then gently shaken (100 rpm) for about 11 to 14 hr at 37°C.

Identification:
Plates were labeled with indelible ink to identify the test article, the strain, the dose level, and the presence or absence of the metabolic activation system.

Metabolic Activation:
Supplier: Molecular Toxicology, Inc., Annapolis, MD
Description: Uninduced hamster liver homogenate preparation (S-9); 29.3 mg protein/mL
Animal strain: Syrian Golden hamsters

Modified Metabolic Activation Mixture:
The modified cofactor mixture with 30% hamster liver S-9 consisted of:
0.09 M sodium phosphate buffer, pH 7.4
20 mM glucose-6-phosphate
2.8 units of glucose-6-phosphate dehydrogenase per mL
3.6 mM NADP
2 mM NADH
2 mM FMN
7.2 mM MgCl2
29.7 mM KCl

Standard Metabolic Activation Mixture:
The standard cofactor mixture with 10% hamster liver S-9 consisted of:
0.1 M sodium phosphate buffer, pH 7.4
5 mM glucose-6-phosphate
4 mM NADP
8 mM MgCl2
33 mM KCl

Experimental Procedure:
To a sterile 13 x 100-mm test tube placed in a 30°C heating block were added:
(1) 0.5 mL of metabolic activation mixture or buffer
(2) 0.1 mL of indicator organisms (about 10E8 bacteria)
(3) appropriate amount of the test article.

The tubes were gently agitated and allowed to incubate for 30 min. Then 2 mL of top agar was added to each tube. This mixture was stirred gently and poured onto plates containing about 25 mL of minimal glucose agar. After the top agar solidified, the plates were incubated at - 37°C for about 48 hr. The histidine-independent revertant colonies were counted after the incubation period; however, if the plates could not be evaluated immediately, they were refrigeratewd at -4°C until they could be counted.

Concurrent sterility and solvent controls were performed with each experiment. Sterility controls included separately plating out the test article, metabolic activation mixture, and buffer. Solvent controls were performed for the positive controls and consisted of top agar, bacteria, metabolic activation mixture or buffer, and 50 uL DMSO, the solvent used to dissolve the positive control substances. The solvent control for the test article, referred to as the zero dose, consisted of top agar, bacteria, metabolic activation mixture or buffer, and the solvent/diluent for the test article.

Evaluation criteria:

Data Collection:
Control of Bias. Bias was controlled by collecting data with an automated colony counter when possible.

Colony Counting. The revertant colonies were counted using an automated colony counter. When accurate counts could not be obtained (e.g., because of precipitation on the plates), the colonies were counted manually using an electric probe colony counter.

Evaluation of Data:
Criteria for Valid Assay: An experiment is considered invalid and is repeated when solvent controls are not within historical limits, when positive control mutagens do not elicit a positive response, or when there are less than three nontoxic dose levels. When experimental plates and sterility control plates indicate gross contamination, the results are not considered valid and the experiment is repeated. In addition, whenever experiments are performed, the strains are analyzed to confirm their genetic markers and the presence of the plasmid. If anomalies exist, the experiment is repeated.

Criteria for Interpretation: The following criteria were used as guidelines for the interpretation of the data; however, the conclusions of the study were based upon the Study Director's evaluation and interpretation of the data.

Positive: A test article is considered a mutagen when a reproducible and statistically significant (p < 0.01) increase in revertants is observed at one or more dose levels. A statistically significant (p < 0.01) dose-related increase in the number of revertants will also be considered a positive response.

Negative: A test article is considered a nonmutagen when the values for the dose levels are not reproducible or significant or when there is no statistically significant dose-related increase in the number of revertants.

Inconclusive: When a test article cannot be identified clearly as a mutagen or nonmutagen, the results are classified as inconclusive.

Statistics:

Statistical Methods: (1) Means and standard deviation were calculated from the individual plate counts; (2) Levene's test (Levene, 1960) was performed to determine if a significant difference exists among treatment variances; (3) treatments were compared with controls by using a one-tailed Dunnett's t-test (Dunnett, 1980) and within-levels pooled variance; and (4) evaluation of dose-relatedness for all treatments was made by regression analysis (Draper and Smith, 1981) of revertant counts versus the log of the concentrations (to allow inclusion of the zero dose, 1 was added to the dose before calculating the log). The significance of the regression was tested using a t-statistic. The statistical analyses were performed using the SAS analysis system, version 6.11, running on a Pentium II computer.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other: A noticeable decrease in the background lawn on all the plates was observed in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

other: A noticeable decrease in the background lawn on all the plates was observed in the absence of metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

All dose levels tested.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Positive at 5μg/plate and above without metabolic activation and at 50μg/plate and above with metabolic activation.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Range-finding Experiment:
Automate Red B (Nonvolatiles) was determined to be soluble at 100 mg/mL in acetone; however, for ease of delivery to the tubes the working solution was further diluted to 50 mg/mL. An initial range-finding experiment was performed with strain TA98 at dose levels of 10, 50, 100, 500, 1000, and 5000 μg/plate (100 μL) in the presence and absence of a reductive metabolic activation system containing 30% hamster liver S-9. A slight increase in the number of revertants was observed in the absence of metabolic activation. The tester strain was inadvertently not added to the tubes in the presence of metabolic activation; thus, no counts were obtained from this portion of the experiment. A noticeable decrease in the background lawn was observed on all test and solvent control plates without metabolic activation which may infer some degree of cytotoxicity caused by acetone. Precipitate was noted on plates at dose levels >= 50 μg/plate in the absence of metabolic activation and at dose levels >=500 μg/plate in the presence of metabolic activation. The bottom agar was stained a yellowish color turning to a purplish color as dose level increased. Both precipitate and staining of agar did not interfere with plate evaluation. In the repeat range-finding experiment the dosing volume was decreased to 50 μL, in an effort to reduce the possible toxic effect of acetone, and dose levels of 10, 50, 100, 250, 500, 1000, and 2500 μg/plate were used in both the presence and absence of the reductive metabolic activation system containing 30% S-9. A noticeable increase in the number of revertants was observed in the presence of metabolic activation and a slight increase was observed in the absence of activation. No cytoxicity was observed in the presence and absence of metabolic activation. Precipitate was noted on plates at dose levels 50 μg/plate in the absence of metabolic activation and at dose levels 100 μg/plate in the presence of metabolic activation. Agar and colonies were stained a yellowish color turning to a purplish color as dose level increased. Both precipitate and staining of agar did not interfere with plate evaluation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation TA98 only
positive without metabolic activation TA98 only
negative with metabolic activation TA1535, TA1537 and TA100
negative without metabolic activation TA1535, TA1537 and TA100

Automate Red B (Nonvolatiles) was found to be reproducibly mutagenic to strain TA98 in the presence and absence of the reductive metabolic activation system.

Executive summary:

Based on the results of the range-finding experiments, the first experiment for mutagenicity was conducted with all four tester strains at dose levels of 0.5, 1, 5, 10, 50, and 100 μg/plate (50 uL) in the presence and absence of the reductive metabolic activation system (30% S-9). The slight increases in the number of revertants seen with strain TA1535 at 5 μg/plate and with strain TA98 at dose levels >= 5 μg/plate in the absence of metabolic activation were statistically significant (p<0.01) by Dunnett's test and only statistically significant by regression analysis with TA98. The slight increase in the number of revertants seen with TA1537 in the presence of metabolic activation was statistically significant only by regression analysis. The increase in the number of revertants seen with TA98 in the presence of reductive metabolic activation system at dose levels 50 μg/plate was statistically significant by Dunnett's test and by regression analysis. No other statistically significant differences were observed. A noticeable decrease in the background lawn on all the plates was observed for strains TA1535 and TA1537 in the absence of metabolic activation. No other signs of cytotoxicity were seen. Precipitate which interfered with machine counts was observed at dose levels >=5 μg/plate in the absence of metabolic activation. These plates were counted manually.

The second experiment for mutagenicity was conducted at dose levels dose levels of 0.5, 1, 5, 10, 50, 100, and 500 μg/plate (50 uL) in the presence and absence of the reductive metabolic activation system (30% S-9). A slight increase in the number of revertants was seen with strain TA98 in the absence of metabolic activation was statistically significant (p < 0.01) by Dunnett's test and statistically significant by regression analysis. In the presence of reductive metabolic activation system, statistically significant increases were seen with TA1537, TA98, and TA100 by regression analysis; however only TA98 was also statistically significant by Dunnett's test. The counts for TA100 were well within the historical value for the strain. No other statistically significant differences were observed. Again, a noticeable decrease in the background lawn on all the plates was observed for strains TA1535 and TA1537 in the absence of metabolic activation. No other signs of cytotoxicity were seen. Precipitate which interfered with machine counts was again observed at dose levels >= 5 μg/plate in the absence of metabolic activation and therefore these plates were counted manually.

Although slight increases that were statistically significant were seen with TA1535 without metabolic activation, TA1537 in the presence of metabolic activation, and TA100 with metabolic activation they were well within their historical range and too slight to be conclusively attributed to mutagenicity.

Automate Red B (Nonvolatiles) was found to be reproducibly mutagenic to strain TA98 in the presence and absence of the reductive metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A peripheral blood mouse micronucleus assay is available. However it is noted that this assay was performed on a commercial product containing a solvent (xylene and ethylbenzene) and the protocol did not include the measurement of body temperature.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study was conducted similar to a guideline study and performed in accordance with GLP; exact details of test material (certificate of analysis, Characterisation) are not included in the report. Test material is commercial product not pure substance

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

The test material appears to be the commercial product and thus contains a solvent (Ethylbenzene and xylene) at approximately 35% of the composition.

Species:

mouse

Strain:

Swiss Webster

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animal specification:
Swiss-Webster mice, 123 males and 123 females, born on May 16, 1989, were received by the SRI Laboratory Animal Medicine Department (LAMD) from Charles River Breeding Laboratories, Inc. (Colony POl) on June 14, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 16.5 to 19.3 g and 14.2 to 16.1 g, respectively, at the time the animals were received. These animals were used for the range-finding study conducted the week of June 25, 1989. swiss-Webster mice, 120 males and 119 females, born on July 18, 1989, were received on August 16, 1989. The weights of 10 male and 10 female mice selected randomly ranged from 13.0 to 14.7 g and 12.0 to 13.2 g, respectively, at the time the animals were received. These animals were used for the definitive study conducted the week of August 27, 1989.

Test System Identification:
The animals were randomized and uniquely identified by ear punch. Cards attached to the outside of each cage contained the study number, test group and subgroup number, dose, and the ear-punch numbers of the animals housed in that cage.

Supplier:
Charles River Breeding Laboratories, Inc. [Colony POl] Shaver Road, Portage, MI 49081

Quarantine:
Mice received on June 14, 1989, and August 16, 1989, were quarantined for seven days and were released on June 21, 1989, and August 23, 1989, respectively. No sign or evidence of significant clinical disease was observed at any time during the quarantine periods or during the study. No notable gross pathologies were found in either 12 male or 12 female mice that were necropsied during the two quarantine periods. Mice not used for this particular study were assigned to concurrently conducted studies within the same project.

Animal Room Environmental Conditions:
Rooms: Building L, Rooms W110 and W107.
Temperature range: 60.8 to 84°F.
Humidity range: 38 to 81%.
Light cycle: 12 hours light/12 hours dark.
Cage specification: Mice were housed no more than 10/cage during quarantine and 5/cage during test period in polycarbonate cages containing hardwood-chip bedding.

Food and water Supply:
Food: Purina certified Rodent Chow #5002 ad libitum. Ralston Purina Co., st. Louis, MO. Lot Nos. APR5891C, MAR30891A, MAY23892B, MAY23892A.
Water: Purified tap water ad libitum via automatic watering system. Water-purity analysis on file, currently in Building 203/45.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): The total volume of test suspension administered per kilogram of body weight was 10 ml.

Negative Controls:
Name: Corn oil.
Lot no.: FEB1490L.
storage conditions: stored at 4°C.
Received: December 20, 1988.
Expiration: December 20, 1989.
Supplier: Mazola
Mission Supermarkets, Best Foods
CPC International, Inc.
Englewood Cliffs, NJ 07632.
Disposition: Retained as vehicle control.

Name: Corn oil.
Lot no.: L12590.
Storage conditions: Stored at 4°C.
Received: July 28, 1989.
Expiration: July 28, 1990.
Supplier: Springfield
PW Supermarkets
Certified Grocers of Calif., LTD.
Los Angeles, CA 90040
Disposition: Retained as vehicle control.

Details on exposure:

Test Article:
Name: Automate Red B.
Lot no.: 7111-2829.
MATERIALS
Purity: Sponsor assumes responsibility.
Physical state at room temperature: Dark red liquid.
Stability: Sponsor assumes responsibility.
Storage conditions: Stored in a secondary lightproof container at room temperature.
Received: June 15, 1989.
Disposition: Return to Sponsor when Final Report is issued.

Treatment:
The route of dosing, the method of administration, and the frequency of administration of the test articles were chosen (in consultation with the Sponsor) to maximize exposure of the animal to the test chemical. The test articles were prepared immediately prior to dosing.

A preliminary dose-range assay was performed to determine the appropriate dose levels for the definitive micronucleus study. Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three/group) with Automate Red B at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW).

For the definitive assay, the test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, 5000 mg/kg BW to 5 males and 5 females per dose group. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B. Five males also received the positive control Benzene at a dose of 500 mg/kg BW.

Duration of treatment / exposure:

For the definitive assay, the test chemical was suspended in corn oil and administered by gavage at 0, 1200, 2500, 5000 mg/kg BW to 5 males and 5 females per dose group. The total volume of test suspension administered per kilogram of body weight was 10 mI. Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B. Five males also received the positive control Benzene at a dose of 500 mg/kg BW.

Frequency of treatment:

Mice were dosed with Automate Red B for four consecutive days.

Post exposure period:

Mice were dosed with Automate Red B for four consecutive days and sacrificed approximately 24 hours after the final dose of Automate Red B.

Dose / conc.:

1 200 mg/kg bw/day (actual dose received)

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Five/sex/dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control: Benzene (500 mg/kg in corn oil) was the positive control. The purpose of the positive control is to ensure proper conduct of the assay procedures. The positive control agent was administered only to 5 male mice

Name: Benzene (99+% pure, 500 mg/kg BW).
Lot no.: 03825EV.
Received: October 10, 1988.
Expiration: October 10, 1998.
Storage conditions: Stored at room temperature.
Supplier: Aldrich Chemical
940 W. st. Paul Avenue
Milwaukee, WI 53233
Disposition: Retained as positive control.

Tissues and cell types examined:

Peripheral Blood Micronucleus Assay:
Blood samples were obtained by pricking the ventral tail vessel with a 25-gauge needle and drawing 2-3 ul of blood into a capillary tube. The sample was transferred to a clean microscope slide, spread, air-dried, fixed in absolute methanol for 5 minutes, and stored until staining. Three slides were prepared from each animal. Immediately prior to scoring, one of the three coded slides from the test animal was stained with acridine orange (Hayashi et al., 1983).

Details of tissue and slide preparation:

Data Collection:
Slides for micronucleus evaluation were coded using random letter codes generated by an IBM PC computer program. Slide labels were printed directly from the computer. Slides were coded by an individual not involved in the microscopic evaluation.

cytological Analysis:
Blood smears were evaluated Two parameters were determined: RNA-positive erythrocytes among using epifluorescence microscopy. (1) the number of micronucleated a total of 1000 RNA-positive erythrocytes per animal, which provides an index of chromosomal damage, and (2) the number of RNA-positive erythrocytes among 5000 erythrocytes per animal, which provides an index of cytotoxicity to the nucleated erythrocyte precursors. The criteria for micronuclei are those described by Schmid (1976), with the additional requirement that they exhibit the fluorescent characteristic of the staining combination (i.e., bright yellow in the case of acridine orange stain). The ratio of RNA-containing erythrocytes to mature erythrocytes (RBC) was based on the number of RNA-positive cells among approximately 5000 erythrocytes. Data from a given slide were entered directly into an IBM PC computer data file while scoring. After analyses were completed, the slides were decoded and the data were summarized using a decoding program on the IBM PC.

Evaluation criteria:

Criteria for a Valid Assay:
The data from this assay were considered acceptable if the frequency of micronucleated cells in the vehicle control group was within the normal historical range, if the positive control article resulted in a statistically significant elevation in the incidence of micronucleated cells, and if there were a minimum of three surviving animals of each sex with a percentage of RNA-positive erythrocytes greater than or equal to 15% of the control value.

Interpretation of Response:
Positive: The test article is considered unequivocally positive if the incidence of micronucleated RNA-containing erythrocytes is significantly higher than in the vehicle control group (p < 0.05) in two different dose groups. A positive dose-related increase in the incidence of micronucleated cells will also be considered a positive response.

Negative: The test article is considered unequivocally negative if the criteria for a positive or inconclusive response are not met.

Inconclusive: The results of this assay will be considered inconclusive if there is reason to believe that the concentrations of the test article selected for evaluation were inappropriate (e.g., excessive toxicity) or if a statistically significant elevation in micronucleated RNA-containing erythrocytes is observed in only one treatment group and the dose-response trend is not significant.

Statistics:

Statistical Tests Employed:
Data from each sex were analyzed both separately and combined, unless a statistically significant sex difference was observed between the vehicle control groups. The frequency of micronucleated RNA-containing erythrocytes among RNA-positive erythrocytes (i.e., the frequency of micronucleated PCES) and the percentage of RNApositive erythrocytes among total erythrocytes were calculated for each animal. The statistical significance of differences in the percentage of RNA-positive erythrocytes among groups was evaluated using the Kruskall-Wallace analysis of variance on ranks. The micronucleus frequency data were analyzed using the CochranArmitage test for trend in binomial proportions, to determine if a significant dose-response relationship was present, and the normal test for equality of proportions, to determine if individual dose groups were statistically elevated above controls. These tests and their rationale are discussed in the ASTM Standard Guide for Conduct of Micronucleus Assays in Mammalian Bone Marrow Erythrocytes (ASTM Committee, 1988).

Sex:

female

Genotoxicity:

negative

Remarks:

All dose levels.

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Sex:

male

Genotoxicity:

negative

Remarks:

1200 and 2500 mg/kg/ BW

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

positive

Remarks:

5000 mg/kg BW

Toxicity:

not specified

Remarks:

No toxicity observed in the preliminary study.

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control was also negative control.

Positive controls validity:

valid

Additional information on results:

Preliminary Study Results:
A preliminary dose-range assay was performed to determine the appropriate dose levels for the definitive micronucleus study. Male and female swiss-Webster mice were given a single dose of the test article by oral intubation (gavage) on Days 1, 2, 3, and 4 and were sacrificed on Day 5. Mice, weighed individually, were dosed (three/group) with Automate Red B at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW). No notable clinical signs were observed, and no animals died as a result of Automate Red B administration. Treatment with the above doses produced polychromatic erythrocyte to red blood cell (PCE/RBC) ratios of 1.30, 1.23, 1.39, 1.54, 1.42, and 0.91%, respectively, in males and 1.57, 1.27, 1.43, 1.29, 1.20, and 1.17%, respectively, in females. The high dose of Automate Red B was over 50% of the control PCE ratio (0.91% vs 1.30%, males; 1.17% vs 1.57%, females) and thus selected as the high dose for the definitive assay. No other statistical evaluations of the range-finding data were deemed necessary.

Definitive Study Results:
Doses of 0, 1200, 2500, and 5000 mg/kg BW of Automate Red B in males produced PCE/RBC values of 1.68, 1.80, 1.75, and 1.75%, respectively. In females, the same doses produced PCE/RBC values of 1.58, 1.75, 1.80, and 1.57%, respectively. There was no apparent decrease in the PCE/RBC ratios; therefore, no additional statistical methods were used to evaluate this parameter.
In male mice, doses of 1200, 2500, or 5000 mg/kg BW of Automate Red B yielded 0.16, 0.22, and 0.52% PCE with MN, respectively, compared with a vehicle control value of 0.20%. In females, the same doses yielded 0.12, 0.20, and 0.16% PCE with MN, respectively, compared with a vehicle control value of 0.26%. In contrast, benzene yielded 3.90% in male mice. The increase in micronucleus frequency in male mice was statistically significant at the high dose, but not at the mid and low doses. The Cochran-Armitage trend test indicated a positive trend for a dose-response relationship; however, this effect is clearly produced by the increase at the high dose.
Because a positive response was observed only at the high dose in male mice, slides from the control and high-dose groups for male mice treated in the range-finding assay (three per group) were rescored for micronuclei to confirm the positive response. This evaluation yielded 0.26 and 0.66% micronucleated PCE for doses of 0 and 5000 mg/kg BW, respectively, a statistically significant elevation.

Conclusions:

Interpretation of results (migrated information): positive/Equivocal
The peripheral erythrocyte micronucleus (MN) assay was used to evaluate the clastogenic potential of Automate Red B. On the basis of the results, it is concluded that Automate Red B induces MN in polychromatic erythrocytes from swiss-Webster mice under the conditions of this assay. The response induced is relatively weak. Benzene produced roughly a 20-fold elevation in micronucleus frequency over controls at a dose of 500 mg/kg. In contrast, Automate Red B produced approximately a 2.5-fold elevation at 5000 mg/kg, making it approximately 80-fold less potent than benzene.

Executive summary:

SRI International assessed the ability of Automate Red B to induce micronuclei (MN) in peripheral erythrocytes following oral administration to male and female swiss-Webster mice. This study was conducted in compliance with the Good Laboratory Practice regulations proposed on December 28, 1987, by the United States Environmental Protection Agency Toxic Substances Control Act

(TSCA; 40 CFR 792). The laboratory work was begun on June 26, 1989, and completed on October 12, 1989. The study was initiated on June 19, 1989, and will be completed when the Final Report is issued.

A range-finding assay was performed to determine the doses used for this study. Mice were dosed, three per group, on Day 1 with Automate Red B suspended in corn oil at 0, 300, 600, 1200, 2500, or 5000 mg/kg body weight (BW) for four consecutive days. Surviving animals were sacrificed approximately 24 hours after the final dose. The number of RNA-positive erythrocytes (polychromatic erythrocytes, PCE) in a field of 5000 red blood cells (RBC) was determined. Only slight suppression of the PCE/RBC ratio was observed at 5000 mg/kg BW in males, and no suppression was observed in females at the same dose. Therefore, the doses selected for the definitive assay were 1200, 2500, and 5000 mg/kg BW. The positive control selected for the definitive assay was benzene (500 mgjkg).

Mice treated with the vehicle control in the definitive assay yielded a MN frequency of 0.20% in males and 0.26% in females. Doses of 1200, 2500, and 5000 mg/kg BW of Automate Red B yielded 0.16, 0.22, and 0.52%, respectively, in males and 0.12, 0.20, and 0.16%, respectively, in females. In contrast, the positive control, benzene, yielded 3.90% PCE with MN in male mice. The increase in MN frequency in male mice was statistically significant at the high dose, but not at the mid and low doses. Slides from the control and

high-dose groups for male mice treated in the range-finding assay evaluation yielded 0.26 and 0.66% micronucleated PCE for doses of 0 and 5000 mg/kg BW, respectively, a statistically significant elevation.

Based on the data obtained from the micronucleus assay, we conclude that Automate Red B induces an increase in micronuclei in peripheral erythrocytes.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Additional information

This substance has been tested for bacterial mutagenicity in several Ames assays. Different lots were used for each assay and the strains tested, methodology and metabolic system varied across the assays. In the 6 assays performed between 1983 and 1984, 5 strains of Salmonella were assessed: TA1535, TA1537, TA1538, TA98, and TA100. Of these assays, two were positive in strains TA 1538 and 98 in the presence of metabolic activation. The remaining assays were negative. These negative assays used a range of dose concentrations (as the top dose) between 5000 and 10000 micrograms per plate and utilised either induced rat liver S9 or uninduced hamster liver S9. One of these negative assays (1984) was conducted using the Prival Modification which is designed to improve the prediction of this assay for azo compounds by increasing the reductive metabolism conditions in the assay.

None of these assays would conform to the current guideline for assessing bacterial mutagenicity due to the absence of the E.coli strain (WP2 uvrA). The only assay performed in accordance with the current guideline is the 1990 assay which included E. coli WP2 uvrA and Salmonella t. strains TA1535, TA1537, TA98, and TA100. This assay was positive in the presence of S9 in strains TA 1537, 98 and 100.

In a final assay, performed in 1999 using a pre-incubation methodology, only 4 strains of Salmonella were assessed, TA 1535, 1537, 98 and 100 and uninduced hamster liver S9 was the metabolic activation system. This assay was positive in only one strain, TA 98 in the presence of S9.

There are therefore 4 out of 8 bacterial mutagenicity assays providing a positive response in the presence of metabolic activitation for at least 1 of the strains, with the majority identifying a positive response in TA 98 and 1538 (frame shift mutations). It is interesting that the assay incorporating the prival modification failed to identify a positive response given the azo-nature of the test material.

It is not possible to provide a definitive reason for some of the mutagenicity assays producing a negative result and others positive. Different lots of test material were used in each assay and there are no detailed analytical information for these lots to identify exact composition and perhaps identify differences. Given that the production of this dye involves the reaction of aromatic amines it is possible that residual amines could have accounted for differences in mutagenic response. It is also possible that residual solvent (ethyl benzene, xylene or even solvent napthalene) could have accounted for the difference in activity, given that some of the lots were also more liquid than others with a range of physical states (and colour intensities) reported for the different assays. However, if one assumes that there was a general consistency in the test materials used in the different assays it is appropriate to conclude that this substance appears to be positive for bacterial mutagenicity in the presence of metabolic activation.

With respect to clastogenicity, in an in vitro chromosome aberration assay using cultured human lymphocytes, the test material is stated as being 99% pure (dark red solid) was tested up to the limit of solubility (in acetone) in the presence and absence of metabolism (rat liver S9). This assay was negative for clastogenicity. In vivo, there is a mouse peripheral erythrocyte micronucleus assay. This assay used a test material that appears to be a commercial product (containing xylene and ethylbenzene solvent) and therefore it is posible that these solvents have impacted the results.

The treatment in this study was 4 consecutive days. For this treatment schedule, the recommended maximum dose level is 2000 mkd in the current testing guideline (OECD TG 474, 2014). The doses in the study 2500 and 5000 mkd possibly exceeded the limit dose, even though the purity was not available. 

 

It is known that some organic solvents can induce micronuclei in erythrocytes in vivo. For instance, this study used benzene as the positive control chemical. The solvent to make the test material a “dark red liquid” was not available in the report but likely Ethylbenzene and xylene. There is a possibility that the observed weak increase in micronucleated erythrocytes in the 5000 mkd male mice was resulted from the solvent in the test although it is noted that studies on ethylbenzene and xylenes do not demonstrate positive results in the micronucleus assay in vivo. 

 

At least 4000 immature erythrocytes per animal should be scored for the incidence of micronucleated immature erythrocytes, according the current testing guideline (OECD TG 474, 2014). Only 1000 PCEs per animal were scored in this study. Given the low background rate (~2/1000) of micronucleated PCE in the study, the incidence of micronucleated PCE in 1000 cells may not stand well for the actual level. The observed weak positive increase has a high possibility to be random. 

 

It is known that body temperature changes can cause micronuclei in erythrocytes (Asanami etc., 1997; Asanami etc., 1998). The body temperature of the animals after test material administration was not monitored in the study. The possibility that the observed weak increase in the incidence of micronucleated PCE resulted from the body temperature change could not be excluded. 

 

Therefore, the lack of test material purity and formulation, the dose selection, the number of cells scored for micronuclei, and the unavailability of body temperature information invalidated this study. The effects of Automate Red B on the incidence of micronucleated erythrocytes in mice need to be re-evaluated following the current test guidelines. 

 

Proposal for further work:

Due to the positive bacterial mutagenicity and questionable findings in the in vivo micronucleus assay it is proposed to perform a combined Comet and micronucleus assay in vivo to further clarify the in vivo mutagenic potential of this substance. Further details can be found in the test proposal record

Justification for classification or non-classification

Although positive in vitro bacterial mutagenicity data are available for this substance, the available in vivo mutagenicity/clastogenicity data are currently insufficient to draw a conclusion regarding potential for mutagenicity. No classification is proposed at this time, and this will be updated once proposed testing is complete.