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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 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

Referenceopen allclose all

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
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Samples of MeNENA were uncapped and placed in a desiccator (with desiccant) for approx. 24 hours prior to use, to remove the added water.
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:
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 100
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 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:
no cytotoxicity
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 MeNENA 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 (2.6-fold) and without S9 (1.6-fold).
-Confirmatory assay: statistically significant, dose-dependent increases in revertant frequencies were observed in strains TA1535 (13-fold) and TA100 (1.4-fold) with S9, and in strain TA1535 without S9 (2.0-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.

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 Me-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: Me-NENA
DOSE LEVEL (µg/plate) S9 TA1535 TA1537 TA1538 TA98 TA100
167 (-) 14 (6) 9 (0) 5 (3) 18 (1) 85 (17)
500 (-) 17 (4) 8 (2) 3 (2) 17 (7) 100 (22)
1670 (-) 17 (5) 8 (2) 4 (2) 17 (3) 102 (24)
5000 (-) 21 (3) 10 (5) 4 (3) 20 (7) 100 (11)
7500 (-) 20 (5) 5 (1) 4 (1) 18 (3) 88 (22)
10000 (-) 24 (3) 7 (1) 3 (2) 18 (2) 99 (9)
               
167 (+) 17 (2) 12 (6) 14 (5) 30 (6) 103 (13)
500 (+) 21 (8) 14 (9) 13 (2) 22 (5) 121 (10)
1670 (+) 32 * (9) 9 (3) 11 (6) 27 (2) 107 (15)
5000 (+) 38 * (9) 5 (2) 14 (3) 23 (6) 121 (7)
7500 (+) 41 * (3) 8 (4) 14 (2) 26 (4) 122 (25)
10000 (+) 37 * (11) 6 (6) 8 (4) 24 (4) 97 (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 3. Confirmatory Mutation Assay on Me-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: Me-NENA
DOSE LEVEL (µg/plate) S9 TA1535 TA1537 TA1538 TA98 TA100
167 (-) 8 (4) 4 (2) 3 (2) 18 (3) 113 (24)
500 (-) 9 (4) 3 (2) 1 (0) 11 (4) 110(22)
1670 (-) 10 (4) 6 (3) 2 (1) 15 (2)  115 (3)
5000 (-) 14 (1) 4 (2) 2 (2) 16 (2) 108 (15)
7500 (-) 16 (4) 5 (3) 3 (3) 13 (1) 110 (16)
10000 (-) 23 * (12) 3 (2) 2 (2) 14 (6) 106 (24)
               
167 (+) 15 (3) 5 (1) 15 (1) 32 (9) 138 (7)
500 (+) 34 *(13) 6 (3) 8 (4) 24 (13) 154 (16)
1670 (+) 63 *(32) 4 (1) 9 (4) 26 (10) 183 (31)
5000 (+) 109 *(20) 6 (5) 11 (3) 17 (10) 182 (4)
7500 (+) 116 * (43) 4 (1) 10 (4) 21 (8) 178 (37)
10000 (+) 66 * (26) 7 (2) 11 (8) 23 (6) 168 (19)

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.

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 Me-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 (2.6-fold) and without S9 (1.6-fold). 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 (13-fold) and TA100 (1.4-fold) with S9, and in strain TA1535 without S9 (2.0-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
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Samples of MeNENA were uncapped and placed in a desiccator (with desiccant) for approx. 24 hours prior to use, to remove the added water.
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:
- With and without S9: 50, 100, 500, 1000, 2500 and 5000 μg/mL.
In a preliminary cytotoxicity test, the test substance produced a slightly toxic effect at the two highest doses with S-9 mix, indicated by a 35% and 32% increase in the Average proliferation time (APT), respectively.
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:
negative
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: Prior to coding, the slides were screened and there were enough scorable metaphases in M2 division at all dose levels evaluated except for 5000 µg/mL with S-9 mix. Therefore, the following doses were coded for analysis: 50, 500, 1000, 2500 and 5000 µg/mL without S-9 mix and at 50, 100, 500, 1000 and 2500 µg/mL with S-9 mix.

RANGE-FINDING/SCREENING STUDIES (if applicable):
MeNENA 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, MeNENA produced a slightly toxic effect at the two highest doses with S-9 mix, indicated by a 35% and 32% increase in APT, respectively. Based on these findings, MeNENA was evaluated in the SCE assay at doses of 50, 100, 500, 1000, 2500 and 5000 µ/mL with and without 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 : MeNENA produced dose dependent increases in the frequency of SCE at all doses evaluated with S-9 mix, with a 5-fold increase over the negative control at the high dose, 2500 µg/mL. See table below.
- Statistical analysis: MeNENA induced statistically significant increases over the negative control, DMSO, at p ≤ 0.01 at all doses evaluated with S-9 mix. In the absence of S-9, MeNENA induced a statistically significant increase (p≤0.05) in SCEfrequency only at the high dose, 5000 µg/mL. However, this increase was not two-fold over the negative control which was the criterion for biological significance. See table below.


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 288 8.08
DMSO (1%) 0 - At-treatment 437 8.88
MeNENA 5 - At-treatment 443 9.05
MeNENA 25 - At-treatment 434 9.09
MeNENA 50 - At-treatment 440 9.09
MeNENA 100 - At-treatment 441 9.11
MeNENA 250 - At-treatment 429 9.11
MeNENA 500 - At-treatment 431 9.05
MeNENA 750 - At-treatment 430 8.98
MeNENA 1000 - At-treatment 397 8.97
MeNENA 2500 - At-treatment 441 8.96
MeNENA 5000 - At-treatment 403 8.95
Untreated 0 - Post-treatment NA 7.09
DMSO (1%) 0 - Post-treatment NA 7.15
MeNENA 5 - Post-treatment NA 7.34
MeNENA 25 - Post-treatment NA 7.43
MeNENA 50 - Post-treatment NA 7.53
MeNENA 100 - Post-treatment NA 7.54
MeNENA 250 - Post-treatment NA 7.55
MeNENA 500 - Post-treatment NA 7.54
MeNENA 750 - Post-treatment NA 7.58
MeNENA 1000 - Post-treatment NA 7.60
MeNENA 2500 - Post-treatment NA 7.59
MeNENA 5000 - Post-treatment NA 7.65

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 0 0 88 12 0 2.06 13.59 0.00
DMSO (1%) 0 - 100 1 3 85 11 0 2.03 13.79 --
MeNENA 5 - 100 0 3 91 6 0 2.02 13.86 0.51
MeNENA 25 - 100 2 15 78 5 0 1.93 14.51 5.22
MeNENA 50 - 100 2 14 83 1 0 1.92 14.58 5.73
MeNENA 100 - 100 5 26 68 1 0 1.83 15.30 10.95
MeNENA 250 - 100 4 23 71 2 0 1.86 15.05 9.14
MeNENA 500 - 100 4 13 82 1 0 1.90 14.74 6.89
MeNENA 750 - 100 8 24 66 2 0 1.81 15.47 12.18
MeNENA 1000 - 100 4 14 78 4 0 1.91 14.66 6.31
MeNENA 2500 - 100 0 5 92 3 0 1.99 14.07 2.03
MeNENA 5000 - 100 2 22 76 0 0 1.87 14.97 8.56
Untreated 0 + 100 0 3 73 24 0 2.11 13.27 0.00
DMSO (1%) 0 + 100 0 5 75 20 0 2.08 13.46 --
MeNENA 5 + 100 0 13 81 6 0 1.97 14.21 5.57
MeNENA 25 + 100 3 25 72 0 0 1.85 15.14 12.48
MeNENA 50 + 100 2 23 73 2 0 1.88 14.89 10.62
MeNENA 100 + 100 3 40 57 0 0 1.77 15.82 17.53
MeNENA 250 + 100 4 40 56 0 0 1.76 15.91 18.20
MeNENA 500 + 100 6 49 45 0 0 1.70 16.47 22.36
MeNENA 750 + 100 2 25 73 0 0 1.86 15.05 11.81
MeNENA 1000 + 100 0 33 67 0 0 1.84 15.22 13.08
MeNENA 2500 + 100 18 56 26 0 0 1.54 18.18 35.07
MeNENA 5000 + 100 13 58 29 0 0 1.58 17.72 31.65

Time in BrdUrd = 28 Hours.

1 - % APT increase is based on a comparison of each dose level to the solvent control.

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 4 7 124 60 5 2.14 13.55 --
DMSO 1% - 200 3 3 151 42 1 2.09 13.88 --
MeNENA 50 - 200 7 6 142 40 5 2.08 13.94 0.43
MeNENA 500 - 200 6 6 150 38 0 2.05 14.15 1.95
MeNENA 1000 - 200 2 0 162 35 1 2.08 13.94 0.43
MeNENA 2500 - 200 2 7 127 62 2 2.14 13.55 --
MeNENA 5000 - 200 1 3 133 59 4 2.16 13.43 --
EMS 124 - 200 4 5 122 68 1 2.14 13.55 --
Untreated 0 + 200 2 7 122 69 0 2.15 13.48 --
DMSO 1% + 200 7 9 121 61 2 2.11 13.74 --
MeNENA 50 + 200 6 11 152 31 0 2.02 14.36 4.51
MeNENA 100 + 200 5 7 177 11 0 1.99 14.57 6.04
MeNENA 500 + 200 9 24 164 3 0 1.90 15.26 11.06
MeNENA 1000 + 200 5 29 164 2 0 1.91 15.18 10.48
MeNENA 2500 + 200 16 37 145 2 0 1.83 15.85 15.36
DEN 100 + 200 6 12 157 25 0 2.00 14.50 5.53

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 5-26 700 991 0.71 14.000 ± 4.764
DMSO 1% - 50 3-24 689 995 0.69 13.780 ± 4.171
MeNENA 50 - 50 8-20 685 995 0.69 13.700 ± 3.125
MeNENA 500 - 50 5-26 721 998 0.72 14.420 ± 5.167
MeNENA 1000 - 50 9-23 745 993 0.75 14.900 ± 3.448
MeNENA 2500 - 50 5-23 716 982 0.73 14.320 ± 4.727
MeNENA 5000 - 50 7-31 788 998 0.79 15.760 ± 5.575*
EMS 124 - 50 20-55 1712 995 1.72 34.240 ± 8.277**
Untreated 0 + 50 7-31 730 996 0.73 14.600 ± 4.806
DMSO 1% + 50 8-34 806 997 0.81 16.120 ± 4.547
MeNENA 50 + 50 30-75 2418 998 2.42 48.360 ± 11.480**
MeNENA 100 + 50 39-91 3155 1002 3.15 63.100 ± 12.477**
MeNENA 500 + 50 53-107 4016 1001 4.01 80.320 ± 11.089**
MeNENA 1000 + 50 49-125 3834 994 3.86 76.680 ± 16.669**
MeNENA 2500 + 50 55-124 4099 989 4.14 81.980 ± 15.837**
DEN 100 + 50 11-48 1289 993 1.30 25.780 ± 8.200**

1 Met = Metaphases

*, ** Denotes a statistically significanct increase at p≤0.05, p≤0.01, respectively.

Conclusions:
The test substance was found positive in the in vitro SCE assay in CHO cells.
Executive summary:

The test substance was evaluated in an 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. The test substance produced a slightly toxic effect at the two highest doses with S-9 mix, indicated by a 35% and 32% increase in the average proliferation time (APT), respectively. Based on these findings, the test substance was evaluated in the SCE assay in duplicate cultures (50 metaphases) at doses of 50, 100, 500, 1000, 2500 and 5000 µg/mL with and without 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. The dose of 5000 µg/mL with S-9 mix was not analysed because there were not enough scorable metaphases. 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 a statistically significant increase (p ≤ 0.05) in SCE frequency only at the high dose, 5000 µg/mL without S-9 but < 2-fold over the negative control and thus considered without biological significance. However, the test substance induced a statistically significant dose-related increases (p ≤ 0.01) in SCE frequencies at all doses evaluated with S-9 mix, with a 5-fold increase over the negative control at the high dose evaluated, 2500 µg/mL. Results of untreated, solvent and positive controls were considered valid. Based on these results it is concluded that the test substance was statistically and biologically positive under the conditions, and according to the criteria, of the test protocol.

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.