Registration Dossier
Registration Dossier
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EC number: 689-188-9 | CAS number: 149343-84-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Genetic toxicity in vitro - Ames Assay
Non-mutagentic with and without metabolic activation in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- .
Genetic toxicity in vitro - Chromosome Aberration
JPR Blue 100 was shown to be non-clastogenic to human lymphocytes in vitro.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 7 April 1993 to 17 June 1993
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- OECD Guidelines for the Testing of Chemicals, Protocol No. 471
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- Method B14 in Commission Directive 92/69/EEC
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- histidine and tryptophan
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Additional strain / cell type characteristics:
- not specified
- Species / strain / cell type:
- E. coli WP2 uvr A
- Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary Toxicity Study: 0, 312.5, 625, 1250, 2500, 5000 μg/plate
Experiment 1: 0, 8.0, 40, 200, 1000, 5000 μg/plate
Experiment 2: 0, 312.5, 625, 1250, 2500, 5000 μg/plate
Appropriate dose levels used in the main study set following the preliminary toxicity study and in accordance with the test guidelines. - Vehicle / solvent:
- JPR Blue 100 was accurately weighed (with an allowance made for purity) and dissolved in sterile distilled water (lot number 301005 108 Exp. 2/96) and appropriate dilutions made.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 9-aminoacridine
- N-ethyl-N-nitro-N-nitrosoguanidine
- benzo(a)pyrene
- other: 2-Aminoanthracene (2AA)
- Details on test system and experimental conditions:
- Microsomal Enzyme Fraction
Lot No. Aro. S9/10/05/93, prepared on 10/05/93 and Aro. S9/23/04/ 93, prepared on 23/04/93, were obtained from the British Industrial Biological Research Association on 11/ 05/93. They were prepared from the livers of male Sprague-Dawley rats weighing ~ 200 g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation.
The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
S9 Mix and Agar
The S9 Mix was prepared at 4 °C as follows:
S9: 5.0 ml
1.65 M KCl / 0.4 M MgCl2: 1.0 ml
0.1 M Glucose-6-phosphate: 2.5 ml
0.1 M NADP: 2.0 ml
0.2 M Sodium phosphate buffer (pH 7.4): 25.0 ml
Sterile distilled water: 14.5 ml
Top agar was prepared using 0.6% Difco Bacto agar (lot number 801679 10/ 96) and 0.5% sodium chloride. For the Salmonella strains 5 ml of 1.0 mM histidine/1.0 mM biotin solution was added to each 100 ml of top agar and for the Escherichia coli strain 5 ml of 1.0 mM tryptophan was added to each 100 ml of top agar. Base agar plates were prepared using 1.2% Oxoid Agar Technical No.3 (lot number 225 62262 7/97 experiment 1 and lot number 336 66640 10/97 experiment 2) with Vogel-Bonner Medium E and 20 mg/ ml 0-glucose.
Test Procedure
a) Preliminary Toxicity Study
In order to select appropriate dose levels for use in the main study, a preliminary test was carried out to determine the toxicity of the test material to the tester organisms. 0.1 ml of bacterial suspension (TA100 or WP2uvrA-), 2 ml of molten, trace histidine or tryptophan supplemented media (histidine/ biotin or tryptophan and top agar), 0.1 ml of test solution and 0.5 ml of phosphate buffer were overlayed onto sterile plates of Vogel -Bonner minimal agar (30 ml / plate). Five doses of the test material and a solvent control (sterile distilled water) were tested in duplicate. After 48 hours incubation at 37 °C the plates were scored for revertant colonies and examined for a thinning of the background lawn.
b) Mutation Study
EXPERIMENT 1
Five concentrations of the test material were assayed in triplicate against each tester strain using the direct plate incorporation method in accordance with the standard methods for mutagenicity tests using bacteria.
Test Material and Negative Controls
0.1 ml aliquots of one of the bacterial suspensions were dispensed into sets of sterile test tubes followed by 2.0 ml of molten trace histidine (or tryptophan in the case of WP2uvrA-) supplemented top agar at 45 °C. These sets comprised two test tubes for each bacterial tester strain. 0.1 ml of the appropriately diluted test material or negative control solution was also added to each of the two tubes, followed by either 0.5 ml of the S9 liver microsome mix or 0.5 ml of pH 7.4 buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material.
Positive Controls
i) Without Activation
0.1 ml of one of the positive control solutions (ENNG, 9AA or 4NQO) was added to a test tube containing 2.0 ml of molten, trace histidine or tryptophan supplemented top agar and 0.1 ml of the appropriate bacterial suspension. Finally, 0.5 ml of pH 7.4 buffer was added to the tube, the contents mixed and poured onto agar plates. This procedure was then repeated in triplicate, for each of the positive controls.
ii) With Activation
0.1 ml of 2AA or BP solution was added to a test tube containing 2.0 ml of molten, trace histidine or tryptophan supplemented top agar and 0.1 ml of one of the test bacterial suspensions. Finally, 0.5 ml of S9 mix was added to the test tube, the contents mixed and poured onto an agar plate. This procedure was then repeated, in triplicate, for each tester strain.
The plates were incubated at 37 °C for 48 hours and the number of revertant colonies counted.
EXPERIMENT 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions in triplicate. - Rationale for test conditions:
- In accordance with test guidelines
- Evaluation criteria:
- For a substance to be considered positive in this test system, it should have induced a dose-related and significant increase in mutation rate (of at least twice the spontaneous reversion rate) in one or more strains of bacteria in the presence and/or absence of the S9 microsomal enzymes in both experiments at sub-toxic dose levels. If the two experiments give conflicting results then a third experiment may be used to confirm the correct response. To be considered negative the number of induced revertants compared to spontaneous revertants should be less than twofold at each dose level employed, the intervals of which should be between 2 and 5 fold and extend to the limits imposed by toxicity or solubility or up to the maximum recommended dose of 5000 μg/plate.
In this case the limiting factor was the maximum recommended dose. - Statistics:
- Not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary Toxicity Study
The dose range of JPR Blue 100 used in the preliminary toxicity study was 0, 312 .5, 625, 1250, 2500 and 5000 μg/plate. JPR Blue 100 was non-toxic in the strains of bacteria used (TA100 and WP2uvrA-).
Mutation Study
The results of the checks for characteristics, viability and spontaneous reversion rate for each tester strain were all found to be satisfactory.
No toxicity was exhibited to any of the strains of bacteria used.
No significant increases in the numbers of revertant colonies of bacteria were recorded for any of the strains of bacteria used, at any dose level, either with or without metabolic activation.
The positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory. - Conclusions:
- The test material, JPR Blue 100, was found to be non-mutagenic under the conditions of this test.
- Executive summary:
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with JPR Blue 100 by the Ames plate incorporation method at five dose levels, in triplicate both with and without the addition of a rat liver homogenate metabolizing system at 10% in standard co-factors. This method conforms to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including MITI, MHW, MOL and MAFF. It also meets the requirements of the OECD, EEC and USA, EPA (TSCA) guidelines. The dose range was determined in a preliminary toxicity assay and was 8 to 5000μg/platein the first experiment. The experiment was repeated on a separate day using different cultures of the bacterial strains and fresh chemical formulations. In this case the dose range of JPR Blue 100 was 312.5 to 5000μg/plate.
The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.
All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system.
JPR Blue 100 caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. JPR Blue 100 was therefore tested up to the maximum recommended dose level of 5000μg/ plate.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of JPR Blue 100, either with or without metabolic activation. JPR Blue 100 was found to be non-mutagenic under the conditions of this test.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 5 July 1993 to 10 January 1994
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- OECO Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test"
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- Method B10 of Commission Directive 92/ 69/EEC
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: in vitro mammalian chromosome aberration test (migrated information)
- Specific details on test material used for the study:
- No further details specified in the study report.
- Target gene:
- structural and numerical aberrations in chromosomes of exposed cells.
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- Cells
Sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability.
The volunteer had not been exposed to high levels of radiation, hazardous chemicals and had not recently suffered from a viral infection. The cell cycle time for each donor is determined on a regular basis. The cell cycle time for the donor used in this study is approximately 17 hours.
Cell Culture
Cells were grown in Eagle's Minimal Essential Medium (MEM), supplemented with 15% foetal calf serum (FCS) and antibiotics, at 37 °C with 5% CO2 in air. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 Mix
- Test concentrations with justification for top dose:
- Experiment 1: 0, 312.5, 625, 1250, 2500, 5000 μg/ml
Experiment 2: 0, 156.25, 312.5, 625, 1250, 2500, 5000 μg/ml
The dose range for evaluation for each treatment condition was selected, on the basis of toxicity, from a series of 8 dose levels (39 to 5000 μg/ml). - Vehicle / solvent:
- JPR blue 100 was accurately weighed and dissolved in MEM and appropriate dilutions made.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- Microsomal Enzyme Fraction
Lot No. Aro. S9/02/07/93, prepared on 02/07/93 and Lot No. Aro. S9/09/07/93, prepared on 09/07/93 were obtained from the British Industrial Biological Research Association on 06/ 07/ 93 and 13/07/93 and were used in Experiment 1 and Experiment 2 respectively.
They were prepared from the livers of male Sprague-Dawley rats weighing ~200 g. These had received a single i.p. injection of Aroclor 1254 at 500 mg/kg, 5 days before S9 preparation. The S9 was stored at -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34.
Culture Conditions - Experiment 1
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components in sterile plastic flasks for each dose level.
- 9.0 ml MEM, 15% (FCS)
- 0.1 ml Li-heparin
- 0.1 ml phytohaemagglutinin-M
- 0.75 - 0.50 ml heparinised whole blood
After 48 hours incubation at 37 °C, 5% CO2 in humidified air, the cells of the with S9 cultures were centrifuged at approximately 1000 rpm for 5 minutes, the culture medium removed and reserved and replaced with 8.0 ml of MEM. 1 ml of a solution of JPR Blue 100 was added in order to give a final concentration of 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 μg/ml in both the with and without S9 cultures. 1 ml of 10% S9 in standard co-factors was added to the with S9 cultures. All the cultures were then returned to the incubator.
Positive control cultures were treated with 500 μg/ml ethyl methanesulphonate in the absence of S9 and 25 μg/ml cyclophosphamide in the presence of S9. Negative control cultures were treated with the test material vehicle (MEM).
After 4 hours of treatment at 37 °C with S9 the cultures were centrifuged for 5 minutes, the treatment medium removed by suction and replaced with an 8 ml wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 16 hours.
Culture Conditions - Experiment 2
These were as above except that further cultures of lymphocytes were harvested 44 hours after the initiation of treatment as well as a 20-hour harvest in Experiment 1. Positive controls were evaluated only in the 20-hour harvest cultures.
Cell Harvest
Mitosis was arrested by addition of demecolcine (0.1 ug/ml) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged (1000-1300 rpm for five minutes), the culture medium drawn off and discarded, and the cells resuspended in 8 ml 0.075M KCl. After fifteen minutes (including five minutes centrifugation}, all but approximately 2.0 ml of hypotonic solution was drawn off and discarded. The cells were resuspended and then fixed by dropping the KCl suspension into 3.0 ml fresh methanol / glacial acetic acid (3:1 v/v). The fixative was changed at least twice and the cells stored at 4 °C for at least four hours to ensure complete fixation.
Preparation of Metaphase Spreads
The lymphocytes were resuspended in 3.0 ml of fresh fixative before centrifugation and resuspension in 0.5 ml of fixative.
Three or four drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.
Staining
When the slides were dry they were stained in 5% Gurrs Giemsa R66 for 5 minutes, rinsed, dried and mounted in Depex mounting medium.
Coding
After checking that the slide preparations were of good quality, the slides were coded using a code from a computerised random number generator.
Scoring of Chromosome Damage
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, and if the cell had 46 or more chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the UKEMS guidelines for mutagenicity testing.
All chromosome aberrations were checked by a senior cytogeneticist prior to decoding the slides.
Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value. - Rationale for test conditions:
- In accordance with test guidelines.
- Evaluation criteria:
- Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors, these are commonly in the range of 0 to 5% cells with structural aberrations.
A positive response was recorded for a particular treatment if the % cells with aberrations markedly exceeded that seen in the concurrent control , either with or without a clear dose-response relationship.
For modest increases in aberration frequency a dose-response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response. - Statistics:
- The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher's Exact test or Chi-squared test.
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- lymphocytes: human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- Dose Selection
The maximum doses used 5000 and 2500 μg/ml were observed to be non -toxic with S9 cultures and toxic in the without S9 cultures. 5000 μg/ml was therefore selected as the highest dose for slide evaluation with 2500 and 1250 μg/ml as the two lower doses for the with S9 cultures. 1250, 625 and 312.5 μg/ml were selected for the without S9 cultures. In the second experiment, JPR Blue 100 was more toxic than in the first experiment therefore lower dose levels were selected for without S9 groups, 625 μg/ml was selected as the highest dose level for evaluation with 312.5 and 156.25 μg/ml as the two lower doses.
Chromosome Aberration Study
In both Experiment 1 and Experiment 2, with S9, JPR Blue 100 did not induce any significant, dose-related decreases in the mitotic index. However, in the without S9 treatments, in both experiments JPR Blue 100 induced a dose-related decrease in the mitotic index. JPR Blue 100 is therefore considered to be toxic to human lymphocytes in vitro without S9 only.
All the negative control cultures gave values of chromosome aberrations within the expected range. The frequency of aberrations was consistent between the negative control groups, the highest frequency (4.5% cells with aberrations including gaps) being seen in Experiment 2.
The positive control cultures gave significant increases in the frequency of aberrations, indicating that the metabolic activation system was satisfactory and that the test method itself was operating as expected.
JPR Blue 100 was seen to induce no significant, dose-related increases in the frequency of aberrations in any of the treatment groups, either with or without S9.
JPR Blue 100 did not induce a significant increase in the numbers of polyploid cells at any dose-level in any of the treatment cases. 0.5% polyploidy was the highest frequency of polyploid cells seen in either the negative controls or the treatment groups and this is within the expected range . - Conclusions:
- JPR Blue 100 produced no significant increases in the frequency of cells with chromosome aberrations either in the presence or absence of a liver enzyme metabolising system. JPR Blue 100 is therefore considered to be non-clastogenic to human lymphocytes in vitro.
- Executive summary:
Human lymphocytes, treated with JPR Blue 100 were evaluated for chromosome aberrations at four dose levels, in duplicate, together with negative and positive controls. Four treatment conditions were used, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest after 16 and 40 hour expression periods and a 20 and 44 hour continuous exposure in the absence of activation . The dose range for evaluation for each treatment condition was selected, on the basis of toxicity, from a series of 8 dose levels (39 to 5000 μg/ ml).
The method used followed that described in the OECD Guidelines for Testing of Chemicals (1981) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 84/ 449/ EEC.
All negative (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
JPR Blue 100 demonstrated no significant increases in the frequency of cells with aberrations in any of the treatment conditions. JPR Blue 100 was shown to be non-clastogenic to human lymphocytes in vitro.
Referenceopen allclose all
Preliminary Toxicity Study
The mean number of revertant colonies for the toxicity assay were:
Strain |
Dose (μg/plate) |
|||||
0 |
312.5 |
625 |
1250 |
2500 |
5000 |
|
TA100 WP2uvrA- |
158.5 38.5 |
176.5 38.0 |
170.5 35.5 |
175.5 36.0 |
172.5 36.0 |
163.0 33.5 |
KEY TO TABLES OF TEST RESULTS
NOTES:
1. When bacterial growth inhibition is found, the applicable value is marked with an asterisk.
2. The average number of colonies for each concentration is recorded in parenthesis.
3. “Number of revertants” – The observed values and average value are shown in order, beginning with the low concentration of the test substance.
4. The following postfixes are used when required:-
C = contaminated
P = precipitate
X = plate unscorable
ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine
4NQO = 4-Nitroquinoline-1-oxide
9AA = 9-Aminoacridine
2AA = 2-Aminoanthracene
BP = Benzo(a)pyrene
TABLE OF TEST RESULTS: EXPERIMENT 1
NAME OF TEST SUBSTANCE: JPR BLUE 100
WITH (+) OR WITHOUT (-) S9 MIX |
TEST SUBSTANCE CONCENTRATION (μg/plate) |
NUMBER OF REVERTANTS (number of colonies/plate) |
|||||||||
BASE-PAIR SUBSTITUTION TYPE |
FRAMESHIFT TYPE |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
- |
0 |
144 144 166 |
(151.3) |
14 9 13 |
(12.0) |
10 14 9 |
(11.0) |
32 27 24 |
(27.7) |
5 12 9 |
(8.7) |
- |
8.0 |
143 167 167 |
(159.0) |
10 11 9 |
(10.0) |
9 14 13 |
(12.0) |
16 22 26 |
(21.3) |
7 7 6 |
(6.7) |
- |
40 |
145 120 152 |
(139.0) |
10 10 10 |
(10.0) |
11 9 13 |
(11.0) |
26 22 22 |
(23.3) |
7 5 6 |
(6.0) |
- |
200 |
135 166 144 |
(148.3) |
11 14 14 |
(13.0) |
9 12 15 |
(12.0) |
21 24 24 |
(23.0) |
6 6 9 |
(7.0) |
- |
1000 |
145 166 161 |
(157.3) |
13 10 9 |
(10.7) |
13 13 18 |
(14.7) |
28 22 19 |
(23.0) |
8 7 7 |
(7.3) |
- |
5000 |
141 160 163 |
(154.7) |
10 13 10 |
(11.0) |
16 14 13 |
(14.3) |
22 26 20 |
(22.7) |
9 8 5 |
(7.3) |
POSITIVE CONTROL NOT REQUIRING S9 MIX |
NAME |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
CONCENTRATION (μg/plate) |
3.0 |
5.0 |
2.0 |
0.2 |
80.0 |
||||||
- |
NUMBER OF COLONIES/PLATE |
508 531 533 |
(557.3) |
149 152 139 |
(146.7) |
276 254 266 |
(265.3) |
134 129 170 |
(144.3) |
1072 1003 1064 |
(1046.3) |
TABLE OF TEST RESULTS: EXPERIMENT 1 (contd)
NAME OF TEST SUBSTANCE: JPR BLUE 100
WITH (+) OR WITHOUT (-) S9 MIX |
TEST SUBSTANCE CONCENTRATION (μg/plate) |
NUMBER OF REVERTANTS (number of colonies/plate) |
|||||||||
BASE-PAIR SUBSTITUTION TYPE |
FRAMESHIFT TYPE |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
+ |
0 |
156 158 176 |
(163.3) |
11 11 17 |
(13.0) |
11 19 9 |
(13.0) |
27 23 18 |
(22.7) |
9 9 7 |
(8.3) |
+ |
8.0 |
149 162 167 |
(159.3) |
11 16 11 |
(12.7) |
20 15 13 |
(16.0) |
19 22 23 |
(21.3) |
14 9 14 |
(12.3) |
+ |
40 |
145 141 140 |
(142.0) |
15 15 10 |
(13.3) |
16 13 10 |
(13.0) |
25 23 20 |
(22.7) |
6 10 7 |
(7.7) |
+ |
200 |
136 162 133 |
(143.7) |
10 10 16 |
(12.0) |
13 14 13 |
(13.3) |
29 24 24 |
(25.7) |
13 5 10 |
(9.3) |
+ |
1000 |
148 158 161 |
(155.7) |
11 14 14 |
(13.0) |
15 19 16 |
(16.7) |
19 19 25 |
(21.0) |
6 10 9 |
(8.3) |
+ |
5000 |
149 155 171 |
(158.3) |
13 9 13 |
(11.7) |
13 12 17 |
(14.0) |
20 23 23 |
(22.0) |
8 10 8 |
(8.7) |
POSITIVE CONTROL REQUIRING S9 MIX |
NAME |
BP |
2AA |
2AA |
BP |
BP |
|||||
CONCENTRATION (μg/plate) |
5.0 |
2.0 |
10.0 |
5.0 |
5.0 |
||||||
+ |
NUMBER OF COLONIES/PLATE |
401 446 420 |
(422.3) |
183 132 172 |
(162.3) |
210 211 204 |
(208.3) |
181 157 156 |
(164.7) |
99 107 110 |
(105.3) |
TABLE OF TEST RESULT: EXPERIMENT 2
NAME OF TEST SUBSTANCE: JPR BLUE 100
WITH (+) OR WITHOUT (-) S9 MIX |
TEST SUBSTANCE CONCENTRATION (μg/plate) |
NUMBER OF REVERTANTS (number of colonies/plate) |
|||||||||
BASE-PAIR SUBSTITUTION TYPE |
FRAMESHIFT TYPE |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
- |
0 |
179 146 184 |
(169.7) |
10 16 11 |
(12.3) |
26 16 15 |
(19.0) |
20 16 15 |
(17.0) |
9 9 10 |
(9.3) |
- |
312.5 |
175 150 160 |
(161.7) |
15 11 11 |
(12.3) |
15 13 11 |
(13.0) |
15 16 21 |
(17.3) |
9 10 11 |
(10.0) |
- |
625 |
150 170 162 |
(160.7) |
10 12 13 |
(11.7) |
19 18 20 |
(19.0) |
16 15 18 |
(16.3) |
8 9 14 |
(10.3) |
- |
1250 |
145 146 166 |
(152.3) |
11 9 15 |
(11.7) |
19 14 11 |
(14.7) |
17 15 22 |
(18.0) |
9 11 9 |
(9.7) |
- |
2500 |
150 146 171 |
(155.7) |
16 12 16 |
(14.7) |
17 17 15 |
(16.3) |
16 18 21 |
(18.3) |
9 12 11 |
(10.7) |
- |
5000 |
151 169 144 |
(154.7) |
11 14 14 |
(13.0) |
9 15 18 |
(14.0) |
19 14 19 |
(17.3) |
10 7 12 |
(9.7) |
POSITIVE CONTROL NOT REQUIRING S9 MIX |
NAME |
ENNG |
ENNG |
ENNG |
4NQO |
9AA |
|||||
CONCENTRATION (μg/plate) |
3.0 |
5.0 |
2.0 |
0.2 |
80.0 |
||||||
- |
NUMBER OF COLONIES/PLATE |
751 707 709 |
(722.3) |
179 161 136 |
(158.7) |
421 427 462 |
(436.7) |
154 174 153 |
(160.3) |
691 701 755 |
(715.7) |
TABLE OF TEST RESULTS: EXPERIMENT 2 (contd)
NAME OF TEST SUBSTANCE: JPR BLUE 100
WITH (+) OR WITHOUT (-) S9 MIX |
TEST SUBSTANCE CONCENTRATION (μg/plate) |
NUMBER OF REVERTANTS (number of colonies/plate) |
|||||||||
BASE-PAIR SUBSTITUTION TYPE |
FRAMESHIFT TYPE |
||||||||||
TA100 |
TA1535 |
WP2uvrA- |
TA98 |
TA1537 |
|||||||
+ |
0 |
184 174 124 |
(160.7) |
15 13 14 |
(14.0) |
20 16 11 |
(15.7) |
26 23 23 |
(24.0) |
13 14 11 |
(12.7) |
+ |
312.5 |
144 132 165 |
(147.0) |
9 15 15 |
(13.0) |
C 18 13 |
(15.5) |
23 20 19 |
(20.7) |
13 11 8 |
(10.7) |
+ |
625 |
148 137 148 |
(144.3) |
14 11 18 |
(14.3) |
18 19 13 |
(16.7) |
25 23 22 |
(23.3) |
14 7 7 |
(9.3) |
+ |
1250 |
159 149 144 |
(150.7) |
18 19 14 |
(17.0) |
10 16 14 |
(13.3) |
16 17 23 |
(18.7) |
7 8 7 |
(7.3) |
+ |
2500 |
145 161 158 |
(154.7) |
11 15 16 |
(14.0) |
18 18 19 |
(18.3) |
19 24 25 |
(22.7) |
9 11 15 |
(11.7) |
+ |
5000 |
170 155 161 |
(162.0) |
10 14 15 |
(13.0) |
16 14 18 |
(16.0) |
22 24 19 |
(21.7) |
7 8 12 |
(9.0) |
POSITIVE CONTROL REQUIRING S9 MIX |
NAME |
BP |
2AA |
2AA |
BP |
BP |
|||||
CONCENTRATION (μg/plate) |
5.0 |
2.0 |
10.0 |
5.0 |
5.0 |
||||||
+ |
NUMBER OF COLONIES/PLATE |
457 458 395 |
(436.7) |
161 178 161 |
(166.7) |
137 141 133 |
(137.0) |
161 178 187 |
(175.3) |
98 119 120 |
(112.3) |
MITOTIC INDEX
EXPERIMENT 1
TEST MATERIAL: JPR BLUE 100
DOSE LEVEL μg/ml |
WITHOUT S9 |
WITH S9 |
||||||
A |
B |
MEAN |
% OF CONTROL |
A |
B |
MEAN |
% OF CONTROL |
|
0 |
12.75 |
10.25 |
11.50 |
100 |
7.40 |
7.50 |
7.45 |
100 |
312.5 |
6.00 |
6.95 |
6.48 |
56 |
- |
- |
- |
- |
625 |
5.15 |
7.15 |
6.15 |
53 |
7.45 |
5.90 |
6.68 |
90 |
1250 |
3.35 |
4.35 |
3.85 |
33 |
6.70 |
7.05 |
6.88 |
92 |
2500 |
2.90a |
3.90a |
3.40 |
30 |
5.35 |
7.25 |
6.30 |
85 |
5000 |
1.45a |
3.00a |
2.23 |
19 |
6.95 |
7.25 |
7.10 |
95 |
EMS 500 |
3.15 |
4.90 |
4.03 |
35 |
- |
- |
- |
- |
CP 25 |
- |
- |
- |
- |
3.30 |
3.70 |
3.50 |
47 |
a= insufficient good quality metaphase cells for analysis.
MITOTIC INDEX
EXPERIMENT 2 – 20-HOUR HARVETS
TEST MATERIAL: JPR BLUE 100
DOSE LEVEL μg/ml |
WITHOUT S9 |
WITH S9 |
||||||
A |
B |
MEAN |
% OF CONTROL |
A |
B |
MEAN |
% OF CONTROL |
|
0 |
6.10 |
7.95 |
7.03 |
100 |
8.20 |
7.45 |
7.83 |
100 |
156.25 |
4.55 |
5.15 |
4.85 |
69 |
- |
- |
- |
- |
312.5 |
3.15 |
2.75 |
2.95 |
42 |
- |
- |
- |
- |
625 |
2.10 |
3.15 |
2.63 |
37 |
7.00 |
7.00 |
7.00 |
89 |
1250 |
2.40a |
1.55a |
1.98 |
28 |
5.55 |
7.35 |
6.45 |
82 |
2500 |
- |
- |
- |
- |
8.75 |
6.60 |
7.68 |
98 |
5000 |
- |
- |
- |
- |
7.55 |
6.60 |
7.08 |
90 |
EMS 500 |
1.60 |
1.45 |
1.53 |
22 |
- |
- |
- |
- |
CP 25 |
- |
- |
- |
- |
1.35 |
1.05 |
1.20 |
15 |
a= insufficient good quality metaphase cells for analysis.
MITOTIC INDEX
EXPERIMENT 2 – 44-HOUR HARVEST
TEST MATERIAL: JPR BLUE 100
DOSE LEVEL μg/ml |
WITHOUT S9 |
WITH S9 |
||||||
A |
B |
MEAN |
% OF CONTROL |
A |
B |
MEAN |
% OF CONTROL |
|
0 |
9.50 |
7.40 |
8.45 |
100 |
7.25 |
3.20 |
5.35 |
100 |
312.5 |
2.30 |
2.40 |
2.35 |
28 |
- |
- |
- |
- |
625 |
1.25a |
0.60a |
0.93 |
11 |
- |
- |
- |
- |
2500 |
- |
- |
- |
- |
5.40 |
6.55 |
5.98 |
114 |
5000 |
- |
- |
- |
- |
5.35 |
4.85 |
5.10 |
98 |
a= too toxic for metaphase analysis
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 1
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 20 HOURS METABOLIC ACTIVATION: NO
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
1 0 |
1 0 |
0 0 |
0 0 |
0 0 |
0 0 |
2 0 |
1 0 |
2 0 |
1 0 |
TOTAL |
200 |
1 (0.5) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
1 (0.5) |
2 (1.0) |
1 (0.5) |
|
312.5 μg/ml |
A B |
100 100 |
3 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
3 0 |
0 0 |
3 0 |
0 0 |
TOTAL |
200 |
3 (1.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
|
625 μg/ml |
A B |
100 100 |
2 0 |
1 1 |
0 0 |
0 1 |
0 0 |
0 0 |
3 2 |
1 2 |
3 2 |
1 2 |
TOTAL |
200 |
2 (1.0) |
2 (1.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
5 (2.5) |
3 (1.5) |
5 (2.5) |
3 (1.5) |
|
1250 μg/ml |
A B |
100 100 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
1 1 |
0 0 |
1 1 |
0 0 |
TOTAL |
200 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
|
POSITIVE CONTROL EMS 500 μg/ml |
A B
|
50 100 |
11 36 |
16 17 |
2 5 |
3 7 |
0 0 |
0 0 |
32 65 |
21 29 |
22 45 |
18 24 |
TOTAL |
150 |
47 (31.3) |
33 (22.0) |
7 (4.7) |
10 (6.7) |
0 (0.0) |
0 (0.0) |
97 (64.7) |
50 (33.3) |
67*** (44.7) |
42*** (28.0) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
*** = p <0.001
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 1
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 20 HOURS METABOLIC ACTIVATION: YES
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
0 1 |
0 0 |
0 0 |
0 1 |
0 0 |
0 0 |
0 2 |
0 1 |
0 2 |
0 1 |
TOTAL |
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
1 (0.5) |
2 (1.0) |
1 (0.5) |
|
1250 μg/ml |
A B |
100 100 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
TOTAL |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
|
2500 μg/ml |
A B |
100 100 |
0 2 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 2 |
0 0 |
0 2 |
0 0 |
TOTAL |
200 |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
2 (1.0) |
0 (0.0) |
|
5000 μg/ml |
A B |
100 100 |
1 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
1 0 |
0 0 |
1 0 |
0 0 |
TOTAL |
200 |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
|
POSITIVE CONTROL CP 25 μg/ml |
A B
|
100 100 |
3 9 |
2 5 |
0 1 |
4 1 |
0 0 |
0 0 |
9 16 |
6 7 |
7 13 |
5 7 |
TOTAL |
200 |
12 (6.0) |
7 (3.5) |
1 (0.5) |
5 (2.5) |
0 (0.0) |
0 (0.0) |
25 (12.5) |
13 (6.5) |
20*** (10.0) |
12** (6.0) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
*** = p <0.001 ** = p <0.01
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 2
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 20 HOURS METABOLIC ACTIVATION: NO
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
1 3 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
2 4 |
1 1 |
2 3 |
1 1 |
TOTAL |
200 |
4 (2.0) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
6 (3.0) |
2 (1.0) |
5 (2.5) |
2 (1.0) |
|
156.25 μg/ml |
A B |
100 100 |
1 4 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
1 4 |
0 0 |
1 3 |
0 0 |
TOTAL |
200 |
5 (2.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
5 (2.5) |
0 (0.0) |
4 (2.0) |
0 (0.0) |
|
312.5 μg/ml |
A B |
100 100 |
2 1 |
4 0 |
0 0 |
0 0 |
0 0 |
0 0 |
6 1 |
4 0 |
5 1 |
3 0 |
TOTAL |
200 |
3 (1.5) |
4 (2.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
7 (3.5) |
4 (2.0) |
6 (3.0) |
3 (1.5 |
|
625 μg/ml |
A B |
100 100 |
0 4 |
0 1 |
0 0 |
0 2 |
0 0 |
0 0 |
0 7 |
0 3 |
0 6 |
0 2 |
TOTAL |
200 |
4 (2.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
7 (3.5) |
3 (1.5) |
6 (3.0) |
2 (1.0) |
|
POSITIVE CONTROL EMS 500 μg/ml |
A B
|
50 100 |
11 20 |
21 20 |
0 1 |
1 5 |
0 0 |
0 0 |
33 46 |
22 26 |
24 29 |
18 22 |
TOTAL |
150 |
31 (20.7) |
41 (27.3) |
1 (0.7) |
6 (4.0) |
0 (0.0) |
0 (0.0) |
79 (52.7) |
48 (32.0) |
53*** (35.3) |
40*** (26.7) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
*** = p <0.001
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 2
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 20 HOURS METABOLIC ACTIVATION: YES
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
5 2 |
3 0 |
0 0 |
0 0 |
0 0 |
0 0 |
8 2 |
3 0 |
7 2 |
3 0 |
TOTAL |
200 |
7 (3.5) |
3 (1.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
10 (5.0) |
3 (1.5) |
9 (4.5) |
3 (1.5) |
|
1250 μg/ml |
A B |
100 100 |
0 0 |
0 0 |
0 0 |
1 0 |
0 0 |
0 0 |
1 0 |
1 0 |
1 0 |
1 0 |
TOTAL |
200 |
0 (0.0) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
1 (0.5) |
1 (0.5) |
1 (0.5) |
1 (0.5) |
|
2500 μg/ml |
A B |
100 100 |
2 1 |
0 2 |
0 0 |
0 0 |
0 1 |
0 0 |
2 4 |
0 3 |
2 4 |
0 3 |
TOTAL |
200 |
3 (1.5) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
1 (0.0) |
0 (0.0) |
6 (3.0) |
3 (1.5) |
6 (3.0) |
3 (1.5) |
|
500 μg/ml |
A B |
100 100 |
0 3 |
0 0 |
0 0 |
0 0 |
0 0 |
0 0 |
0 3 |
0 0 |
0 3 |
0 0 |
TOTAL |
200 |
3 (1.5) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
|
POSITIVE CONTROL CP 25 μg/ml |
A B
|
100 100 |
10 4 |
21 7 |
1 1 |
4 4 |
0 0 |
0 0 |
36 16 |
26 12 |
16 11 |
15 9 |
TOTAL |
200 |
14 (7.0) |
28 (14.0) |
2 (1.0) |
8 (4.0) |
0 (0.0) |
0 (0.0) |
52 (26.0) |
38 (19.0) |
27* (13.5) |
24*** (12.0) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
* = p <0.05 *** = p <0.001
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 2
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 44 HOURS METABOLIC ACTIVATION: NO
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
3 3 |
1 1 |
0 0 |
0 0 |
0 0 |
0 0 |
4 4 |
1 1 |
4 4 |
1 1 |
TOTAL |
200 |
6 (3.0) |
2 (1.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
0 (0.0) |
8 (4.0) |
2 (1.0) |
8 (4.0) |
2 (1.0) |
|
312.5 μg/ml |
A B |
100 100 |
1 7 |
5 3 |
0 0 |
0 3 |
0 0 |
0 0 |
6 13 |
5 6 |
4 10 |
4 3 |
TOTAL |
200 |
8 (4.0) |
8 (4.0) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
0 (0.0) |
19 (9.5) |
11 (5.5) |
14 (7.0) |
7 (3.5) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
DATA SUMMARY SHEET – RESULTS OF CHROMOSOME ABERRATION ASSAY – EXPERIMENT 2
TEST MATERIAL: JPR BLUE 100 HARVEST TIME: 44 HOURS METABOLIC ACTIVATION: YES
TREATMENT GROUP |
REPLICATE IDENTIFICATION |
NO. OF CELLS SCORED |
TOTAL GAPS |
CHROMATID |
CHROMOSOME |
OTHERS |
TOTAL ABERRATIONS |
ABERRANT CELLS |
||||
BREAKS |
EXCHANGES |
BREAKS |
EXCHANGES |
X |
(+Gaps) |
(-Gaps) |
(+Gaps) |
(-Gaps) |
||||
NEGATIVE CONTROL |
A B |
100 100 |
1 2 |
1 0 |
0 0 |
0 1 |
0 0 |
0 0 |
2 3 |
1 1 |
1 3 |
1 1 |
TOTAL |
200 |
3 (1.5) |
1 (0.5) |
0 (0.0) |
1 (0.5) |
0 (0.0) |
0 (0.0) |
5 (2.5) |
2 (1.0) |
4 (2.0) |
2 (1.0) |
|
5000 μg/ml |
A B |
100 100 |
3 0 |
3 0 |
0 0 |
3 0 |
0 0 |
0 0 |
9 0 |
6 0 |
7 0 |
4 0 |
TOTAL |
200 |
3 (1.5) |
3 (1.5) |
0 (0.0) |
3 (1.5) |
0 (0.0) |
0 (0.0) |
9 (4.5) |
6 (3.0) |
7 (3.5) |
4 (2.0) |
|
X = >10 aberration per cell (not included in total aberrations) Figures in brackets = aberrations per 100 cells
INCIDENCE OF POLYPLOID CELLS (%) IN HUMAN LYMPHOCYTES AFTER TREATMENT WITH JPR BLUE 100
EXPERIMENT 1
DOSE LEVEL μg/ml |
20 HOURS |
|
WITHOUT S9 |
WITH S9 |
|
0 312.5 625 1250 2500 5000 EMS 500 CP 25 |
0.0 0.0 0.0 0.5 - - 0.0 - |
0.0 - - 0.0 0.0 0.0 - 0.0 |
EXPERIMENT 2
DOSE LEVEL μg/ml |
20 HOURS |
44 HOURS |
||
WITHOUT S9 |
WITH S9 |
WITHOUT S9 |
WITH S9 |
|
0 156.25 312.5 625 1250 2500 5000 EMS 500 CP 25 |
0.5 0.5 0.5 0.0 - - - 0.0 - |
0.0 - - - 0.0 0.0 0.0 - 0.0 |
0.0 - 0.0 - - - - - - |
0.0 - - - - - 0.5 - - |
- = not applicable
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Genetic toxicity in vitro - Ames Assay
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA- were treated with JPR Blue 100 by the Ames plate incorporation method at five dose levels, in triplicate both with and without the addition of a rat liver homogenate metabolizing system at 10% in standard co-factors.
The solvent (sterile distilled water) control plates gave counts of revertant colonies within the normal range.
All positive control chemicals produced marked increases in the numbers of revertant colonies, both with and without the metabolising system.
JPR Blue 100 caused no reduction in the growth of the bacterial lawn at any dose level either with or without metabolic activation. JPR Blue 100 was therefore tested up to the maximum recommended dose level of 5000μg/ plate.
No significant increase in the numbers of revertant colonies was recorded for any of the bacterial strains with any dose of JPR Blue 100, either with or without metabolic activation. JPR Blue 100 was found to be non-mutagenic under the conditions of this test.
Genetic toxicity in vitro - Chromosome Aberration
Human lymphocytes, treated with JPR Blue 100 were evaluated for chromosome aberrations at four dose levels, in duplicate, together with negative and positive controls. Four treatment conditions were used, i.e. 4 hours exposure with the addition of an induced rat liver homogenate metabolising system at 10% in standard co-factors with cell harvest after 16 and 40 hour expression periods and a 20 and 44 hour continuous exposure in the absence of activation .
All negative (solvent) controls gave frequencies of cells with aberrations within the range expected for normal human lymphocytes.
All the positive control treatments gave significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.
JPR Blue 100 demonstrated no significant increases in the frequency of cells with aberrations in any of the treatment conditions. JPR Blue 100 was shown to be non-clastogenic to human lymphocytes in vitro.
Justification for classification or non-classification
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