Registration Dossier
Registration Dossier
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EC number: 285-332-6 | CAS number: 85068-73-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro bacteria reverse mutation: Key study: Test method similar to the OECD Guideline 471 with GLP study. The test substance was found positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.
In vitro SCE assay in mammalian cells: Key study: Test method similar to the OECD Guideline 479 with GLP study. The test substance was found positive with and without metabolic activation in the in vitro SCE assay in CHO cells under the conditions, and according to the criteria, of the test protocol.
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:
- 30 September 1991 - 31 October 1991
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- E. coli WP2 or S. typhimurium TA102 were not tested.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine-requiring gene in Salmonella typhimurium.
- 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:
- Type and composition of metabolic activation system:
- source of S9 : male sprague-Dawley rat
- method of preparation of S9 mix: The S9 mixture contained 8mM MgCI2 , 33mM KCl, 4mM NADP, 5mMglucose-6-phosphate, 100mM Na2 HP04 (pH 7.4) and 6% (v/v) Aroclor 1254 induced liver homogenate .
- concentration or volume of S9 mix and S9 in the final culture medium: Cultures treated in the presence of S9 contained 0.5 mL of the S9 mixture.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): S9 mix was evaluated for sterility. - Test concentrations with justification for top dose:
- 167, 500, 1670, 5000, 7500 and 10000 μg/plate.
In a preliminary toxicity test performed with strains TA1538 and TA100 (-S9), no cytotoxicity was found at doses up to 5000 μg/plate. In addition, the test article was freely soluble at all doses evaluated. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO), Lot #902873, supplied by Fisher Scientific (Fairlawn, NJ).
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- TA100/-S9 and TA1535/-S9 (10 μg/plate)
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537/-S9 (150 μg/plate)
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- TA98/-S9 and TA1538/-S9 (5 μg/plate)
- Positive controls:
- yes
- Positive control substance:
- other: 2-anthramine
- Remarks:
- TA 1535, TA 1537, TA 98 and TA 100 and 1538 / +S9 (2.5 μg/plate)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : triplicate
- Number of independent experiments : one initial experiment and one confirmatory assay (in case of positive results in the first assay)
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1-2 x 109 cells/mL.
- Test substance added in agar (plate incorporation). Without metabolic activation, 0.1 ml tester strain, 0.1 ml of the appropriate concentration of the test article or solvent and 2 ml of molten top agar (supplemented with 0.5mM histidine/0.5mM biotin) were mixed. For the assay with metabolic activation, 0.5 ml of the S9 mixture was also added.
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition.
- Any supplementary information relevant to cytotoxicity: Toxicity of the substance was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 at doses of 0.0 (solvent control), 50.0, 167, 500, 1670 and 5000 μg /plate in the absence of S9. The toxicity was determined by evaluating the growth of the background lawn and/or frequency of spontaneous revertants.
METHODS FOR MEASUREMENTS OF GENOTOXICIY
Following incubation for 48 hours, revertant colonies were enumerated on an Artek electronic colony counter interfaced with an IBM PC/AT computer for data acquisition.
Solvent and positive controls were scored first, and test article treated cultures were scored only if the average negative control values were within historical ranges (x ± 2SD;see table below).
- Evaluation criteria:
- A positive result was defined as a statistically significant, dose-dependent increase in the number of histidine-independent revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value.
The result was considered equivocal when the test article did not induce a statistically significant, dose-dependent increase in revertant frequency, but did induce a revertant frequency at one dose level that was two-fold the spontaneous control value.
A negative result was defined as the absence of a statistically significant or dose-dependent increase in the number of histidine-independent revertants. - Statistics:
- Statistical analyses were performed using the program developed by Snee and Irr (1981), with significance established at the 95% confidence limit.
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight toxicity at 10000 µg/plate (-S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight/moderate toxicity from 5000 µg/plate.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight toxicity from 5000 µg/plate.
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- 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 examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- slight/moderate toxicity from 5000 µg/plate (+S9)
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES (if applicable):
Results of the toxicity prescreen indicated EtNENA was not toxic (characterized as normal background lawn growth) to strains TA1538 and TA100 at doses of 50.0, 167, 500, 1670 and 5000 µg/plate in the absence of S9. In addition, the test article was freely soluble at all doses evaluated.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : see table below.
For all test methods and criteria for data analysis and interpretation:
- First assay: statistically significant, dose-dependent increases in revertant frequencies were observed in strain TA1535 with (19-fold) and without S9 (2.6-fold), and in strains TA98 (1.6-fold) and TA100 (3.1-fold) with S9.
-Confirmatory assay: statistically significant, dose-dependent increases in revertant frequencies were observed in strains TA1535 (20-fold) and TA100 (1.3-fold) with S9, and in strain TA1535 without S9 (2.1-fold).
Ames test:
- Signs of toxicity: see table below
- Mean number of revertant colonies per plate and standard deviation: see table below.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Not reported
- Negative (solvent/vehicle) historical control data: see table below. - Conclusions:
- The test substance was found positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.
- Executive summary:
The test substance Et-NENA was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay following a method similar to OECD Guideline 471 in a GLP study. The assay (plate incorporation method) was carried out in triplicate using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA1538 in the presence and absence of metabolic activation (S9 mixture prepared with 6% (v/v) Aroclor 1254-induced male sprague-Dawley rat liver homogenate). Dimethyl sulfoxide (DMSO) was used as solvent. Toxicity of the substance was first evaluated in a prescreen by treating duplicate cultures of strains TA1538 and TA100 at doses of 0.0 (solvent control), 50.0, 167, 500, 1670 and 5000 μg /plate in the absence of S9. The substance was found no toxic to each strain at all doses tested. In addition, the test substance was freely soluble at all doses evaluated. Based on these findings, the test substance was evaluated in the mutation assay at doses of 0.0 (solvent control), 167, 500, 1670, 5000, 7500 and 10000 µg/plate with and without S9. Triplicate cultures of each strain were evaluated with the solvent (negative control) and the appropriate positive control (sodium azide, 9-aminoacridine and 2-nitrofluorene for assay “without metabolic activation” and 2-anthramine for assay “with metabolic activation”) in the same conditions as that used for the test substance. A positive result was defined as a statistically significant, dose-dependent increase in the number of revertants with at least one dose level inducing a revertant frequency that is two-fold the spontaneous solvent control value. According to the results, statistically significant, dose-dependent increases in revertant frequencies were observed in strain TA1535 with (19-fold) and without S9 (2.6-fold), and in strains TA98 (1.6-fold) and TA100 (3.1-fold) with S9. Thus, a confirmatory assay was conducted in the same conditions with the following results: statistically significant, dose-dependent increases in revertant frequencies were observed in strains TA1535 (20-fold) and TA100 (1.3-fold) with S9, and in strain TA1535 without S9 (2.1-fold). The mean values of revertant colonies of the negative (vehicle) control plates were within the historical control range. Also, positive control values were within acceptable limits. Based on the results of the study, it is concluded that the test item was positive in the Ames/Salmonella Plate Incorporation Assay under the conditions, and according to the criteria, of the test protocol.
- Endpoint:
- in vitro DNA damage and/or repair study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 02 October 1991 - 31 December 1991
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- sister chromatid exchange assay in mammalian cells
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: CHO-K1-BH4, Lot #M7. Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratories P.O. Box Y, Oak Ridge, Tennessee 37830.
For cell lines:
- Cell cycle length, doubling time or proliferation index : 12-14 hours.
- Modal number of chromosomes: 20
MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: F12FCM(5%) medium [Ham's medium F12 (K.C. Biological Co., reconstituted with deionized water, adjusted to pH 7.5, followed by the addition of 1.2 g/l NaHC03) containing 5% heat-inactivated (56°C, 30 min.) fetal bovine serum (K.C. Biological Co.) extensively dialyzed by Pharmakon Research International, Inc.]; 37ºC, 5% C02, ≥90 % humidity. - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- Type and composition of metabolic activation system:
- source of S9: Aroclor 1254 induced rat liver.
- method of preparation of S9 mix: The S9 mixture contained (per ml) 10mM MgCl2, 10mM CaCI2, 30mM KCI, 5mM glucose-6-phosphate, 4mM NADP (disodium salt), 50mM sodium phosphate buffer (pH 7.4) and 0.1 ml of the microsomal preparation containing approximately 34.8 mg protein/mI.
- concentration or volume of S9 mix and S9 in the final culture medium: Cultures treated in the presence of S9 contained 2 mL of the S9 mixture. - Test concentrations with justification for top dose:
- - Without S9: 50, 250, 500, 2000 and 5000 μg/mL.
- With S9: 50, 250, 500, 2000, 4000 and 5000 μg/mL.
In a preliminary cytotoxicity test, the test substance produced a significant dose-related increase in the Average proliferation time, APT (50% over the solvent control) with S-9 mix at the highest dose (5000 μg/mL). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO), Lot #902873, supplied by Fisher Scientific (Fairlawn, NJ).
- Untreated negative controls:
- yes
- Remarks:
- (F12 medium)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (DMSO 1% v/v)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- without metabolic activation, 124 μg/mL (10- 3M).
- Positive controls:
- yes
- Positive control substance:
- other: N-nitrosodiethylamine
- Remarks:
- with metabolic activation, 100 μg/mL (9.8 x 10-4 M)
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration : duplicate.
- Number of independent experiments : 1
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 8 x 105 cells/80 cm2 flask.
- Test substance added in F12 serum free medium.
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 16-24 hr.
- Exposure duration/duration of treatment: 5 hr.
- Harvest time after the end of treatment (sampling/recovery times): 29 hr (incubation with medium F12FCM(5%) and 5μM BrdUrd)
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): colcemid (2 x 10-7 M final concentration), added to cultures for the last 2 hours of the incubation period with F12FCM(5%) and 5μM BrdUrd.
- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): At the end of incubation, cell suspensions were collected by the mitotic shake-off method. Cells were sedimented by centrifugation for approximately 5-10 minutes at 1000 rpm and hypotonic KCI (0.075M) added to swell the cells. Cells were fixed in three washes of methanol: glacial acetic acid (3 parts: 1 part) and slides prepared by standard methods. Staining of slides by the FPG method included: 1.0 μg/ml Hoechst 33258 stain, black light irradiation and 2-3% Giemsa stain. Slides were air-dried and coverslips mounted.
- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): a total of 50 (25 metaphases per culture) well-spread, second division cells containing ± 2 centromeres from the modal number of 20 were scored for each dose level.
- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): SCE were scored as reciprocal alterations in staining pattern along the chromatids of a chromosome. Cells were counted for chromosome number and data were presented as SCE/metaphase and SCE/chromosome. The mean cell cycle (MCC) was based on the ratio of first, second and third division metaphases per metaphases scored. The APT (average proliferation time) was expressed as the ratio of exposure time of a population of cells in BrdUrd to the respective MCC.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cell proliferation kinetics. This biological endpoint estimates the average proliferation time (APT) in which a population of CHO cells has undergone cell divisions in the presence of the thymidine analog, 5-bromo-2' -deoxyuridine (BrdUrd). Any increase in APT over the solvent control was an indication of cytotoxicity. See below more information on the calculations.
- Any supplementary information relevant to cytotoxicity: It has been shown that an increase in osmolality (ion concentrations) and/or non-physiological pH are genotoxic to cultured mammalian cells (Brusick, D., 1986 and Galloway, et al., 1987 and Morita, T., et al., 1989). Therefore, the osmolality and pH of the test article dilutions were evaluated and compared to the negative control, DMSO.
- Evaluation criteria:
- A positive response was based on the ability of the test substance to produce a statistically significant increase in the SCE frequency as compared to the concurrent solvent control. If the t test indicated a statistically positive result at a single dose level only, this was insufficient grounds to regard the test article as positive, although the presence of a dose response in consecutive dose levels would justify retesting, using additional concentrations and/or fixation times (Perry et al.,1984).
SCE results were in addition interpreted with due regard for the biological significance of the data. For biological significance, a two-fold increase in SCE frequency in at least one dose level as compared to the negative control and/or a significant dose-response pattern were considered. - Statistics:
- Duplicate cultures were pooled to make a total of 50 scored metaphases per dose level. The SCE/metaphase data was transformed by a standard square root transformation. A t test on the transformed data compared each dose level against the solvent control.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: There were no significant changes in the pH of the dosing solutions as compared to the solvent control (see table below).
- Data on osmolality: There were no significant changes in osmolality of the dosing solutions as compared to the solvent control (see table below).
- Definition of acceptable cells for analysis: At the time of colcemid addition, the majority of the cells in the 5000 µg/mL cultures with S-9 mix were shrunken, detached and lysed. These cultures were discarded.
RANGE-FINDING/SCREENING STUDIES (if applicable):
EtNENA was initially evaluated in a cytotoxicity test in CHO cells at doses of 5, 25, 50, 100, 250, 500, 750, 1000, 2500 and 5000 µg/mL with and without S-9 mix.
The results of this assay (see table below) indicated that there was no significant increase in APT at any dose evaluated without S-9 mix. However, EtNENA produced a significant dose-related increase in APT (50%) with S-9 mix at the highest dose (5000 µg/mL). Based on these findings, EtNENA was evaluated in the SCE assay at doses of 50, 250, 500, 2000 and 5000 µg/mL without S-9 mix and at doses of 50, 250, 500, 2000, 4000 and 5000 µg/mL with S-9 mix.
STUDY RESULTS
- Concurrent vehicle negative and positive control data : See results in tables below. Vehicle negative controls had ≤18 SCE per metaphase and both positive controls EMS and DEN produced significant increases (p ≤ 0.01) in SCE frequency as compared to the solvent control.
For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : EtNENA induced dose-related increases in SCE frequencies at all doses evaluated with approx. 3.6- to 6.3-fold increases over the negative control, DMSO, with and without S-9 mix, respectively, except for the 50 µg/mL dose without S-9 mix. See table below.
- Statistical analysis; EtNENA induced statistically significant increases over the negative control, DMSO, at p ≤ 0.01 at all doses evaluated with and without S-9 mix except for the 50 µg/mL dose without S-9 mix. See table below.
- Conclusions:
- The test substance was found positive with and without metabolic activation in the in vitro SCE assay in CHO cells.
- Executive summary:
The test substance was evaluated in the in vitro SCE assay, similar to OECD TG 479 and with GLP, to determine its potential to induce an increase in SCE frequency as compared to DMSO, the solvent control, in CHO cells with and without S-9 mixture (S-9 mix prepared with Aroclor 1254-induced male rat liver homogenate). Cytotoxicity was first evaluated utilizing cell proliferation kinetics. The test substance was evaluated in CHO cells at doses of 5, 25, 50, 100, 250, 500, 750, 1000, 2500 and 5000 µg/mL with and without S-9 mix. A significant dose-related increase in the average proliferation time (APT) up to 50% with S-9 mix at the highest dose was obtained. Based on these findings, the test substance was evaluated in the SCE assay in duplicate cultures (50 metaphases) at doses of 50, 250, 500, 2000 and 5000 µg/mL without S-9 mix and at doses of 50, 250, 500, 2000, 4000 and 5000 µg/mL with S-9 mix. After 5 h treatment in F12 serum free medium, the cultures were washed, and fresh medium (F12FCM, 5%) and BrdUrd were added and incubated for 29 h. Colcemid (2 x 10-7M) was added 2 h prior to harvest to each culture to arrest cells in metaphase. The cells were harvested, and slides were prepared and stained for sister chromatid differentiation. Positive controls N-nitrosodiethylamine (DEN) and Ethylmethane Sulfonate (EMS) were used with and without S-9 mix, respectively. Also, an untreated control (F12 medium only) was run in parallel. The test substance induced statistically significant, dose-related increases in SCE frequencies at all doses evaluated with approx. 3.6- to 6.3-fold increases over the solvent control with and without S-9 mix, respectively, except for the 50 µg/mL dose without S-9 mix. Results of untreated, solvent and positive controls were considered valid. It is concluded that the test substance was statistically and biologically positive under the conditions, and according to the criteria, of the test protocol.
Referenceopen allclose all
Table 1. Historical Data - Spontaneous Revertants*
| Strain | S9 | n | Average (± 1SD) |
Range (X± 2SD) |
| TA1535 | - | 173 | 9.65 ± 2.78 | 4.08 - 15.2 |
| + | 176 | 10.3 ± 2.80 | 4.67 - 15.9 | |
| TA1537 | - | 172 | 7.87 ± 2.54 | 2.79 - 12.9 |
| + | 171 | 9.50 ± 2.75 | 4.00 - 15.0 | |
| TA1538 | - | 177 | 5.92 ± 2.41 | l.11 - 10.7 |
| + | 178 | 13.5 ± 3.60 | 6.26 - 20.6 | |
| TA98 | - | 186 | 19.0 ± 4.92 | 9.14 - 28.8 |
| + | 195 | 27.4 ± 6.77 | 13.9 - 40.9 | |
| TA100 | - | 184 | 83.8 ± 15.9 | 52.0 - 116 |
| + | 188 | 96.9 ± 16.0 | 64.8 - 129 |
*January 1, 1990 - September 30, 1991
Table 2. Original Mutation Assay on Et-NENA
| CONTROLS | |||||||
AVERAGE REVERTANTS/PLATE |
|||||||
Solvent controls |
S9 | TA1535 | TA1537 | TA1538 | TA98 | TA100 | |
| DMSO (100µL) | (-) | 15 (4) | 10 (2) | 3 (3) | 21 (6) | 100 (9) | |
| DMSO (100µL) | (+) | 16 (2) | 10 (3) | 14 (2) | 27 (1) | 107 (18) | |
| Positive controls | (µg/plate) | ||||||
| SODIUM AZIDE | 10.0 | (-) | 1356 * (96) | ---(---) | ---(---) | ---(---) | 1312 * (114) |
| 9-AMINOACRIDINE | 150 | (-) | ---(---) | 1279 * (38) | ---(---) | ---(---) | ---(---) |
| 2-NITROFLUORENE | 5.00 | (+) | ---(---) | ---(---) | 471 * (66) | 408 * (44) | ---(---) |
| 2-ANTHRAMINE | 2.50 | (+) | 117 *(24) | 604 * (166) | 1671 * (133) | 2298 * (212) | 2370 * (195) |
TEST ARTICLE: Et-NENA |
|||||||
| DOSE LEVEL (µg/plate) | S9 | TA1535 | TA1537 | TA1538 | TA98 | TA100 | |
| 167 | (-) | 14 (3) | 12 (4) | 6 (3) | 21 (12) | 97 (12) | |
| 500 | (-) | 16 (3) | 12 (4) | 7 * (5) | 22 (3) | 86 (11) | |
| 1670 | (-) | 22 (6) | 7 (3) | 6 * (3) | 23 (7) | 96 (6) | |
| 5000 | (-) | 36 * (5) | 6 (3) | 4 (1) | 18 (2) | 102 (10) a | |
| 7500 | (-) | 39 * (2) | 5 (1) | 4 (3) | 21 (1) | 119 (10) a | |
| 10000 | (-) | 39 * (7) | 6 (1) | 2 (1) | 20 (10) a | 113 (21) a | |
| 167 | (+) | 111 *(50) | 15 (3) | 14 (4) | 44 (4) | 149 (19) | |
| 500 | (+) | 184 * (81) | 12 (5) | 18 (7) | 36 (11) | 253 *(44) | |
| 1670 | (+) | 295 * (78) | 12 (1) | 22 (6) | 43 (4) | 335 * (79) | |
| 5000 | (+) | 223 * (102) | 8 (2) | 16 (5) a | 28 (2) | 251 * (12)a | |
| 7500 | (+) | 149 * (58) | 9 (3) | 17 (7) a/b | 29 (1) | 233 *(12)a | |
| 10000 | (+) | 82 * (8) | 11 (4) | 11 (6) a/b | 32 (9) | 197 *(28)a/b | |
Data reported as: Mean (Standard Deviation).
*Positive response (≥ 2X solvent control value. Only two-fold or greater increases are indicated)
a/b = slight / moderate toxicity.
No precipitate.
Table 3. Confirmatory Mutation Assay on Et-NENA
| CONTROLS | |||||||
AVERAGE REVERTANTS/PLATE |
|||||||
| Solvent controls | S9 | TA1535 | TA1537 | TA1538 | TA98 | TA100 | |
| DMSO (100µL) | (-) | 11 (4) | 9 (7) | 4 (3) | 19 (2) | 114 (3) | |
| DMSO (100µL) | (+) | 9 (4) | 7 (1) | 12 (5) | 24 (3) | 131 (12) | |
| Positive controls | (µg/plate) | ||||||
| SODIUM AZIDE | 10.0 | (-) | 1140 * (62) | ---(---) | ---(---) | ---(---) | 926 * (133) |
| 9-AMINOACRIDINE | 150 | (-) | ---(---) | 1231 * (55) | ---(---) | ---(---) | ---(---) |
| 2-NITROFLUORENE | 5.00 | (+) | ---(---) | ---(---) | 273 * (26) | 287 * (39) | ---(---) |
| 2-ANTHRAMINE | 2.50 | (+) | 137 *(22) | 603 * (63) | 1507 * (130) | 2173 * (128) | 2055 * (239) |
TEST ARTICLE: Et-NENA |
|||||||
| DOSE LEVEL (µg/plate) | S9 | TA1535 | TA1537 | TA1538 | TA98 | TA100 | |
| 167 | (-) | 12 (3) | 5 (2) | 2 (1) | 19 (7) | 120 (22) | |
| 500 | (-) | 15 (2) | 4 (1) | 1 (1) | 19 (8) | 103 (27) | |
| 1670 | (-) | 13 (6) | 4 (2) | 4 (2) | 10 (7) | 133 (17) | |
| 5000 | (-) | 16 (5) | 4 (2) | 2 (3) | 11 (5) | 106 (5) | |
| 7500 | (-) | 19 (1) | 2 (1) | 2 (1) | 16 (1) | 126 (6) | |
| 10000 | (-) | 24 * (5) | 3 (1) | 0 (1) | 13 (5) | 118 (4) | |
| 167 | (+) | 46 *(33) | 5 (2) | 11 (3) | 26 (9) | 143 (29) | |
| 500 | (+) | 128 * (50) | 9 (7) | 11 (3) | 25 (4) | 133 (12) | |
| 1670 | (+) | 172 * (63) | 7 (3) | 15 (2) | 19 (5) | 172 (25) | |
| 5000 | (+) | 153 * (53) | 5 (4) | 7 (1) | 27 (2) | 165 (15) | |
| 7500 | (+) | 128 * (27) | 9 (5) | 5 (2) | 19 (7) | 160 (22) | |
| 10000 | (+) | 82 * (25) | 6 (3) | 2 (1) | 16 (8) | 119 (12) | |
Data reported as: Mean (Standard Deviation).
*Positive response (≥ 2X solvent control value. Only two-fold or greater increases are indicated)
Apparently normal growth all strains/doses +/-S9.
No precipitate.
Table 1. Osmolality and pH in culture Medium of CHO cells
| Compound | Dose (µg/mL) |
S-9 | Phase | Average Osmolality (mOSM/ kg H2O) |
pH |
| Untreated | 0 | - | At-treatment | 297 | 8.56 |
| DMSO (1%) | 0 | - | At-treatment | 441 | 8.60 |
| EtNENA | 5 | - | At-treatment | 400 | 8.61 |
| EtNENA | 25 | - | At-treatment | 340 | 8.66 |
| EtNENA | 50 | - | At-treatment | 461 | 8.66 |
| EtNENA | 100 | - | At-treatment | 439 | 8.64 |
| EtNENA | 250 | - | At-treatment | 4,3 | 8.65 |
| EtNENA | 500 | - | At-treatment | 427 | 8.65 |
| EtNENA | 750 | - | At-treatment | 416 | 8.68 |
| EtNENA | 1000 | - | At-treatment | 426 | 8.68 |
| EtNENA | 2500 | - | At-treatment | 410 | 8.67 |
| EtNENA | 5000 | - | At-treatment | 407 | 8.70 |
| Untreated | 0 | - | Post-treatment | NA | 6.93 |
| DMSO (1%) | 0 | - | Post-treatment | NA | 6.99 |
| EtNENA | 5 | - | Post-treatment | NA | 7.09 |
| EtNENA | 25 | - | Post-treatment | NA | 7.10 |
| EtNENA | 50 | - | Post-treatment | NA | 7.18 |
| EtNENA | 100 | - | Post-treatment | NA | 7.19 |
| EtNENA | 250 | - | Post-treatment | NA | 7.19 |
| EtNENA | 500 | - | Post-treatment | NA | 7.23 |
| EtNENA | 750 | - | Post-treatment | NA | 7.20 |
| EtNENA | 1000 | - | Post-treatment | NA | 7.19 |
| EtNENA | 2500 | - | Post-treatment | NA | 7.26 |
| EtNENA | 5000 | - | Post-treatment | NA | 7.19 |
NA - Not applicable
NOTE: The S-9 mix was added to a set of cultures to test the potential of the test article to be biotransformed by the liver enzymes (cytochrome P-450) into a clastogen. Consequently, the osmolality and pH of the solvent and dosing solutions were only measured in the set of cultures without S-9 mix. The high pH at treatment was due to the buffer capacity (sodium bicarbonate) of the F12SF medium. Once the cultures were placed in 5% C02 incubator, the pH of the medium became equilibrated to a physiologic pH. Therefore, the pH was measured at-and post-treatment times while the osmolality was only measured at treatment time.
Table 2. Cell Proliferation Kinetics Analysis - Cytotoxicity
| Compound | Dose (µg/mL) |
S-9 (±) |
Total nº Metaphases scored | No. of Mitotic Divisions | MCC | APT (hrs) | %APT Increase 1 | ||||
| M1 | M1+ | M2 | M2+ | M3 | |||||||
| Untreated | 0 | - | 100 | 1 | 24 | 75 | 0 | 0 | 1.87 | 14.97 | 4.25 |
| DMSO (1%) | 0 | - | 100 | 1 | 10 | 88 | 1 | 0 | 1.95 | 14.36 | -- |
| EtNENA | 5 | - | 100 | 1 | 11 | 86 | 2 | 0 | 1.95 | 14.36 | 0.00 |
| EtNENA | 25 | - | 100 | 0 | 14 | 82 | 4 | 0 | 1.95 | 14.36 | 0.00 |
| EtNENA | 50 | - | 100 | 2 | 8 | 85 | 5 | 0 | 1.97 | 14.21 | 0.00 |
| EtNENA | 100 | - | 100 | 3 | 7 | 88 | 2 | 0 | 1.95 | 14.36 | 0.00 |
| EtNENA | 250 | - | 100 | 3 | 10 | 85 | 2 | 0 | 1.93 | 14.51 | 1.04 |
| EtNENA | 500 | - | 100 | 0 | 15 | 76 | 9 | 0 | 1.97 | 14.21 | 0.00 |
| EtNENA | 750 | - | 100 | 5 | 15 | 78 | 2 | 0 | 1.89 | 14.81 | 3.13 |
| EtNENA | 1000 | - | 100 | 1 | 10 | 88 | 1 | 0 | 1.95 | 14.36 | 0.00 |
| EtNENA | 2500 | - | 100 | 2 | 5 | 91 | 2 | 0 | 1.97 | 14.21 | 0.00 |
| EtNENA | 5000 | - | 100 | 5 | 12 | 83 | 0 | 0 | 1.89 | 14.81 | 3.13 |
| Untreated | 0 | + | 100 | 1 | 5 | 93 | 1 | 0 | 1.97 | 14.21 | 0.50 |
| DMSO (1%) | 0 | + | 100 | 0 | 9 | 87 | 4 | 0 | 1.98 | 14.14 | -- |
| EtNENA | 5 | + | 100 | 0 | 14 | 84 | 2 | 0 | 1.94 | 14.43 | 2.05 |
| EtNENA | 25 | + | 100 | 5 | 17 | 78 | 0 | 0 | 1.87 | 14.97 | 5.87 |
| EtNENA | 50 | + | 100 | 7 | 22 | 71 | 0 | 0 | 1.82 | 15.38 | 8.77 |
| EtNENA | 100 | + | 100 | 4 | 31 | 65 | 0 | 0 | 1.81 | 15.47 | 9.41 |
| EtNENA | 250 | + | 100 | 15 | 37 | 48 | 0 | 0 | 1.67 | 16.77 | 18.60 |
| EtNENA | 500 | + | 100 | 9 | 45 | 46 | 0 | 0 | 1.69 | 16.57 | 17.19 |
| EtNENA | 750 | + | 100 | 10 | 44 | 46 | 0 | 0 | 1.68 | 16.67 | 17.89 |
| EtNENA | 1000 | + | 100 | 5 | 59 | 36 | 0 | 0 | 1.66 | 16.87 | 19.31 |
| EtNENA | 2500 | + | 100 | 28 | 63 | 9 | 0 | 0 | 1.41 | 19.86 | 40.45 2 |
| EtNENA | 5000 | + | 100 | 42 | 53 | 5 | 0 | 0 | 1.32 | 21.21 | 50.00 2 |
Time in BrdUrd = 28 Hours.
1 - % APT increase is based on a comparison of each dose level to the solvent control.
2 - 40 and 50% increases indicated a significant increases in APT. Generally the highest dose selected for SCE is the dose which increase APT ≤50%.
Table 3. Cell Proliferation Kinetics Analysis
| Compound | Dose (µg/mL) |
S-9 (±) |
Total nº Metaphases scored | No. of Mitotic Divisions | MCC | APT (hrs) | %APT Increase 1 | ||||
| M1 | M1 + | M2 | M2+ | M3 | |||||||
| Untreated | 0 | - | 200 | 0 | 4 | 169 | 24 | 3 | 2.07 | 14.01 | -- |
| DMSO | 1% | - | 200 | 0 | 2 | 172 | 24 | 2 | 2.07 | 14.01 | -- |
| EtNENA | 50 | - | 200 | 3 | 2 | 181 | 140 | 2 | 2 | 14.36 | 2.50 |
| EtNENA | 250 | - | 200 | 0 | 1 | 182 | 16 | 1 | 2.04 | 14.22 | 1.50 |
| EtNENA | 500 | - | 200 | 2 | 5 | 174 | 18 | 1 | 2.03 | 14.29 | 2.00 |
| EtNENA | 2000 | - | 200 | 6 | 5 | 175 | 14 | 0 | 1.99 | 14.57 | 4.00 |
| EtNENA | 5000 | - | 200 | 6 | 8 | 152 | 32 | 2 | 2.04 | 14.22 | 1.50 |
| EMS | 124 | - | 200 | 2 | 3 | 158 | 37 | 0 | 2.08 | 13.94 | -- |
| Untreated | 0 | + | 200 | 3 | 5 | 170 | 22 | 0 | 2.03 | 14.29 | 6.96 |
| DMSO | 1% | + | 200 | 0 | 2 | 129 | 67 | 2 | 2.17 | 13.36 | -- |
| EtNENA | 50 | + | 200 | 7 | 32 | 150 | 11 | 0 | 1.91 | 15.18 | 13.62 |
| EtNENA | 250 | + | 200 | 11 | 29 | 153 | 6 | 1 | 1.89 | 15.34 | 14.82 |
| EtNENA | 500 | + | 200 | 15 | 58 | 123 | 4 | 0 | 1.79 | 16.20 | 21.26 |
| EtNENA | 2000 | + | 200 | 42 | 94 | 64 | 0 | 0 | 1.56 | 18.59 | 39.15 |
| EtNENA | 4000 | + | 200 | 26 | 133 | 41 | 0 | 0 | 1.54 | 18.83 | 40.94 |
| DEN | 100 | + | 200 | 1 | 5 | 166 | 27 | 1 | 2.06 | 14.08 | 5.39 |
Time in BrdUrd = 29 Hours.
1 - % APT increase is based on a comparison of each dose level to the solvent control.
Table 4. In vitro sister Chromatid Exchange in CHO Cells
| Compound | Dose (µg/mL) |
S-9 (±) |
Total nº Metaphases scored | Range of SCE/Met 1 | Total number of SCE's | Total number of chromosomes | SCE/ chromosome |
SCE/Met S.D. |
| Untreated | 0 | - | 50 | 8-25 | 764 | 1001 | 0.76 | 15.280 ± 3.839 |
| DMSO | 1% | - | 50 | 6-27 | 759 | 995 | 0.76 | 15.180 ± 4.685 |
| EtNENA | 50 | - | 50 | 7-26 | 810 | 995 | 0.81 | 16.200 ± 4.932 |
| EtNENA | 250 | - | 50 | 9-29 | 914 | 997 | 0.92 | 18.280 ± 4.928** |
| EtNENA | 500 | - | 50 | 9-36 | 1055 | 999 | 1.06 | 21.100 ± 6.004** |
| EtNENA | 2000 | - | 50 | 15-68 | 1705 | 1000 | 1.71 | 34.100 ± 12.261** |
| EtNENA | 5000 | - | 50 | 23-105 | 2735 | 994 | 2.75 | 54.700 ± 19.925** |
| EMS | 124 | - | 50 | 14-73 | 1933 | 1000 | 1.93 | 38.660 ± 11.070** |
| Untreated | 0 | + | 50 | 7-28 | 830 | 998 | 0.83 | 16.600 ± 4.986 |
| DMSO | 1% | + | 50 | 6-31 | 813 | 996 | 0.82 | 16.260 ± 4.763 |
| EtNENA | 50 | + | 50 | 38-98 | 3030 | 1006 | 3.01 | 60.600 ± 11.925** |
| EtNENA | 250 | + | 50 | 63-121 | 4498 | 997 | 4.51 | 89.960 ± 14.977** |
| EtNENA | 500 | + | 50 | 61-132 | 4496 | 998 | 4.51 | 89.920 ± 16.199** |
| EtNENA | 2000 | + | 50 | 63-110 | 4518 | 992 | 4.55 | 90.360 ± 12.008** |
| EtNENA | 4000 | + | 50 | 75-156 | 5077 | 999 | 5.08 | 101.540 ± 16.314** |
| DEN | 100 | + | 50 | 20-46 | 1535 | 999 | 1.54 | 30.700 ± 6.707** |
1 Met = Metaphases
*, ** Denotes a statistically significanct increase at p≤0.05, p≤0.01, respectively.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (positive)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Justification for classification or non-classification
Based on the available information, further studies are required to conclude on classification of the test substance in accordance with CLP Regulation (EC) no 1272/2008.
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