A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February 22 to April 18, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed following good laboratory practices, and according to OECD Guidelines. The study followed standard protocol and was well documented.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Version / remarks:

Although the study did not specifically specify Guideline 472, the study title and statement of following OECD guideline, points to Guideline 472.

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

87787-81-3

EC Number:

618-070-1

Cas Number:

87787-81-3

IUPAC Name:

87787-81-3

Constituent 2

Reference substance name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

EC Number:

413-800-3

EC Name:

A mixture of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid; dihydrogenated alkyl (C14-C18)amine

Cas Number:

87787-81-3

Molecular formula:

C44H79NO3

IUPAC Name:

2-(dioctadecylcarbamoyl)benzoic acid; 2-(ditetradecylcarbamoyl)benzoic acid; 2-[(propan-2-yloxy)carbonyl]benzoic acid; 2-[octadecyl(tetradecyl)carbamoyl]benzoic acid; 2-octadecyl-2,3-dihydro-1H-isoindole-1,3-dione; 2-tetradecyl-2,3-dihydro-1H-isoindole-1,3-dione; benzene-1,2-dicarboxylic acid; dioctadecylamine; ditetradecylamine; octadecyl(tetradecyl)amine

Constituent 3

Reference substance name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

IUPAC Name:

reaction mass of: N,N-di(hydrogenated alkyl C14-C18)phthalamic acid dihydrogenated alkyl (C14-C18)amine

Constituent 4

Reference substance name:

Stepan TAB-2

IUPAC Name:

Stepan TAB-2

Details on test material:

Test substance Lot No.: 198247 of unspecified purity. Test substance consisted of yellow, wax-like flakes, and was stored at room temperature., protected from exposure to light.

Method

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Study also included S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

See Details on test system and conditions text below. Concentrations employed are: Vehicle only, 100, 333, 1000, 3333 and 10,000µg/plate.

Vehicle / solvent:

acetone

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Migrated to IUCLID6: Also others listed in table in Any other information on materials and Methods section

Details on test system and experimental conditions:

Media and Reagent Preparation:

All water used was sterile, deionized distilled grade water.

On the day of its use, minimal top agar, containing 0.8% agar (w/v) and 0.5% NaCl (w/v) was melted and supplemented with either L-histidine and D-biotin solution to a final concentration of 50 µg/ml or with tryptophan to a final concentration of 11 µg/ml. Top agar not used with S-9 mix was supplemented with 25 ml of sterile water for each 100 ml of minimal top agar.

Bottom agar was Vogel-Bonner minimal medium E (Vogel and Bonner, 1956) containing 1.5% (w/v) agar and supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder).

Nutrient broth was Vogel-Bonner salt solution supplemented with 2.5% (w/v) Oxoid Nutrient Broth No. 2 (dry powder).

Aroclor 1254-induced rat liver S-9 was used as the metabolic activation system. The S-9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg five days prior to sacrifice. The S-9 was batch prepared and stored at ≤-70°C until used. Each bulk preparation of S-9 was assayed for its ability to metabolize 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to Salmonella typhimurium TA100. The microsomal enzyme mixture (S-9 mix) was prepared immediately before its use and contained 5 mM glucose-6-phosphate, 4 mM β-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33mM KCl in a 100 mM phosphate buffer at pH 7.4.

Test System:
Tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535, TA1537, TA1538 and Escherichia coli tester strain WP2 uvrA.

Frozen Permanent Stocks were prepared by growing fresh overnight cultures, adding DMSO (0.09 ml/ml of culture) and freezing away approximately 1.5 ml aliquots in glass vials. Frozen Permanent Stocks were stored at ≤-70°C. Master plates were prepared by streaking each Salmonella tester strain from a frozen permanent onto minimal medium supplemented with histidine (260µgM) biotin (3µgM) and for strains containing the R-factor, ampicillin (25µg/ml). Master plates for E. coli were prepared by streaking onto nutrient bottom agar. Master plates were stored at 4±2°C.

Overnight cultures were prepared by removing a colony of the appropriate tester strain from appropriate the master plate and transferring it to a vessel containing about 50 ml of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at about 125 rpm at 37±2°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of approximately 1-2 x E+009 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

On the day of their use in the mutagenicity assay, all tester strain cultures were checked for the correct genotype. The presence of the rfa wall mutation was confirmed for Salmonella by demonstration of sensitivity to crystal violet. The presence of the pKM101 plasmid was confirmed for Salmonella by demonstration of resistance to ampicilin. Spontaneous reversion frequencies in the vehicle controls that are characteristic of the respective strains were demonstrated by plating 100 µl aliquots of the culture along with the appropriate vehicle on selective media. The acceptable ranges are as follows:
TA98 10 – 50
TA100 80-240
TA1535 5-45
TA1537 3-21
TA1538 5-35
WP2uvrA 10-60

Experimental Design:

The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975). This methodology has been shown to detect many classes of chemical mutagens. The assay was performed in two phases. The first phase, the dose-range finding study, was used to establish the dose range over which the test article would be assayed. Ten dose levels of the test material were plated, one plate per dose, with overnight cultures of TA100 and WP2 uvrA on selective minimal agar in both the presence and absence of rat microsomal enzymes. The second phase, the mutagenicity and confirmatory assays, was used to evaluate the mutagenicity. A minimum of five dose levels of test material along with appropriate vehicle and positive controls were plated with tester strains TA98, TA100, TA1535, TA1537, TA1538 and WP2uvrA in the presence and absence of rat liver microsomal enzymes. All dose levels of test material, vehicle controls and positive controls were plated in triplicate.

Test and Control Materials:

The vehicle used to deliver Stepan TAB-2 to the test system was acetone (certified A.C.S., purchased from Fisher Scientific Company, Silver Spring, MD). The dosing solutions were not adjusted to compensate for the purity of the test material. Aliquots of dosing solutions preparations were not analyzed.

To determine the sterility of the test material, the highest test material dose level used In the mutagenicity assay was plated on selective agar with an aliquot volume equal to that used in the assay. To determine the sterility of the S-9 mix, a 0.5 ml aliquot was plated on selective agar.

Plating and Scoring Procedures:
Each plate was labeled with a code system that identified the test material, test phase, dose level, tester strain and activation.

Test material dilutions were prepared immediately before use. In the absence of S-9 mix, 100µl of tester strain and 100µl of vehicle or test material were added to 2.5 ml of molten selective top agar at 45±2°C. When S-9 mix was required, 500µl of S-9 mix, 100µl of tester strain and 100µl of vehicle or test material were added to 2.0 ml of molten selective top agar at 45±2°C. When plating the positive controls the test article aliquot was replaced by a 50µ aliquot of appropriate positive control. After vortexing the mixture was overlaid onto the surface of 25 ml of minimal bottom agar. After the overlay had solidified the plates were inverted and incubated for approximately 48 hours at 37±2°C. Plates that were not counted immediately following the 48-hour incubation period were stored at 4±2°C until colony counting could be conducted.

The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. This toxicity was scored relative to the vehicle control plate. Revertant colonies for a given tester strain and activation condition, except for the positive controls, were counted either entirely by automated colony counter or entirely by hand. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.

For each replicate plating the mean and standard deviation of the number of revertants per plate were calculated.

Evaluation criteria:

The following criteria must be met for the mutagenicity assay to be considered valid. All Salmonella tester strain cultures must demonstrate the presence of the deep rough mutation (rfa) and cultures of tester strains TA98, and TA100 must demonstrate the presence of the pKM101 plasmid R-factor. All cultures must demonstrate the characteristic mean number of spontaneous revertants in the vehicle control To ensure that appropriate numbers of bacteria are plated, tester strain culture titers must be greater than or equal to 0.6xE+009 cells/ml. The mean of each positive control must exhibit at least a three-fold increase in the number of revertants over the man value of the respective vehicle control. A minimum of three non-toxic dose levels are required to evaluate assay data. A dose level is considered toxic if one or both of the following criteria are met: (1) A > 50% reduction in the mean number of revertants per plate as compared to the mean vehicle control value. This reduction must be accompanied by an abrupt dose-dependent drop in the revertant count. (2) A reduction in the background lawn.

For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test material. Data sets for strains TA1535, TA1537 and TA1538 will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than three times the mean vehicle control value. Data sets for strains TA98, TA100 and WP2uvrA will be judged positive if the increase in mean revertants at the peak of the dose response is equal to or greater than two times the mean vehicle control value.

Statistics:

Averages and standard deviations were determined and tabulated.

Results and discussion

Test results

Species / strain:

other: TA98, TA100, TA1535, TA 1537, TA1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results of the dose range finding study are presented in Tables 1 and 2. The maximum dose tested was 10,000 µg per plate. This dose was delivered to the test system as a suspension in acetone using a plating aliquot of 100µl.

The results of the dose range-finding study of Stepan TAB-2 conducted in the presence and absence of microsomal enzymes indicate that no appreciable toxicity was observed up to 10,000 µg/plate. When no toxicity is observed, the maximum dose to be plated in the mutagenicity assay will be 10,000µg per plate.

The results of the mutagenicity assays were tabulated. These data were generated in Experiments T9784-B1, T9784-B2 and T9784-B3. In Experiment T9784-B1 no positive responses were observed with tester strains TA98, TA1535, TA1537, TA-1538 and WP2uvrA in the presence of microsomal enzymes and with any of the other tester strains in the absence of microsomal enzymes. Due to an unacceptable positive control, tester strain TA100 in the presence of microsomal enzymes was not evaluated but retested in Experiment T9784-B2. Independently, the confirmatory assay was conducted in Experiment T9784-B3.

In Experiment T9784-B2 no positive response was observed with tester strain TA100 in the presence of microsomal enzymes.

In Experiment T9784-B3, the confirmatory assay, no positive responses were observed with any of the tester strains in the presence and absence of microsomal enzymes.

All criteria for a valid study were met as described in the protocol with the exception of the titer values for the cultures of tester strains TA1535. TA1537 and TA1538 used in Experiment T9784-B1. However, since the culture turbidity data and all other strain characterization indicators were acceptable (the vehicle and positive control values and the ampicillin and crystal violet sensitivities), the study director accepted the data generated with these cultures.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Salmonella/E. coli Mutagenicity Assay

Summary of Results

Table 27

 

Test Article Id: Stepan TAB-2 (CFTA Name: Di (hydrogenated) tallow phthalic acid amide)             

Study Number: T9784.501038                    Experiment Nos: B1/B2                                                                                                                                                               

Average Revertants Per Plate ± Standard Deviation

Liver Microsomes: None

Dose(ug)                            TA98                     TA100                   TA1535                TA1537                TA1538           WP2 uvrA

0.0                                         29 ±      4             198 ±   16          10 ±      1             6 ±        4             7 ±        2             11 ±      2

100                                        25 ±      5             206 ±   7             8 ±        2             6 ±        2             7 ±        5            15 ±      4

333                                        26 ±      4             191 ±   9             12 ±      5             10 ±      4             7 ±        3            18 ±      7

1000                                      25 ±      4             184 ±   8             14 ±      2             5 ±        2             6 ±        5            13 ±      1

3333                                      25 ±      2             179 ±   16          13 ±      2              6 ±        2             5 ±        2             17      4

10000                                   27 ±       6            178 ±   16          7 ±        4              8 ±        6             7 ±        2              14 ±     8

Pos                                        129 ±   25           610 ±   23          333 ±   45          23 ±      6             205 ±   2              159 ±   18

 

Liver Microsomes: Rat liver S-9

Dose(ug)                            TA98                     TA100                   TA1535                TA1537                TA1538           WP2 uvrA

0.0                                         27 ±      6             147 ±    5            13 ±      2             6 ±        3             13 ±      5           22 ±      4

100                                        24 ±      5             145 ±   14          13 ±      1             5 ±        5            12 ±      1             16 ±      3

333                                        30 ±       5            164 ±   12          17 ±      2             8 ±        2             10 ±      4            11 ±      1

1000                                      26 ±      1             131 ±   10          16 ±       4            8 ±        2             13 ±      1            16 ±      2

3333                                      19 ±      6             160 ±   24          14 ±      3             8 ±        2             10 ±      4             9 ±        4

10000                                   28 ±      5              146 ±   17          19 ±       3            11 ±      4             20 ±      11          13 ±       5

Pos                                        272 ±   19           458 ±   34           54 ±      24           42 ±      14          367 ±   39           248 ±    48

0.0 = Vehicle plating aliquot of 100 µL

Pos= Positive Control concentrations as specified in Materials and Methods Section

Salmonella/E. coli Mutagenicity Assay

Summary of Results

 

Table 28- P. 779

 

Test Article Id: Stepan TAB-2 (CFTA Name: Di (hydrogenated) tallow phthalic acid amide)             

Study Number: T9784.501038                    Experiment Nos. B3                                                                                                                                               

Average Revertants Per Plate ± Standard Deviation

Liver Microsomes: None

Dose(ug)                            TA98                     TA100                   TA1535                TA1537                TA1538           WP2 uvrA

0.0                                         16 ±      2             113±    12          7 ±        1             6±         3             5 ±        2             16±       4

100                                        20 ±      2             101 ±   7             10 ±      4             5 ±        3             5 ±        1            16 ±      6

333                                        14 ±      3             118 ±   12          10 ±      3             5 ±        2             5 ±        3             17 ±      3

1000                                      10 ±      3             122 ±   2             9 ±        5             5 ±        1             7 ±        3            17 ±      4

3333                                      16 ±      5             114 ±   5             13 ±      4             5 ±        3             3 ±        3           19 ±       9

10000                                   18 ±       2             132 ±   7             12 ±      2             8 ±        1             6 ±        1            24 ±      5

Pos                                        310 ±   21          376 ±   5             312 ±   15          143 ±   10            444 ±   5               58±      17

 

Liver Microsomes: Rat liver S-9

Dose(ug)                            TA98                     TA100                   TA1535                TA1537                TA1538           WP2 uvrA

0.0                                         22±       4             151 ±    6            12 ±      4             7 ±        2             15 ±      2            18 ±      1

100                                        20 ±      5             153 ±   12          14 ±      2             7 ±        2            9 ±        1              15 ±      5

333                                        24 ±       5             129 ±   8             12 ±      3             4 ±        2             14 ±      1           12 ±      1

1000                                      21 ±      3             140 ±   14          12 ±       3             5 ±        1             13 ±      7           14 ±      4

3333                                      22 ±      6             145 ±   3             13 ±      3             5 ±        1             9 ±        3           15 ±      5

10000                                    27 ±      2             150 ±   14           15 ±      5             8 ±        3             11 ±      2           33 ±       9

Pos                                        368 ±   7             498 ±   22           44 ±      8             34 ±      6             460 ±   18          211±    35

0.0 = Vehicle plating aliquot of 100 µL

Pos= Positive Control concentrations as specified in Materials and Methods Section

Applicant's summary and conclusion

Conclusions:

The results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay(Ames Test) and E. coli WP2 uvrA reverse Mutation Assay indicate under the conditions of this study that test substance Stepan TAB-2 showed a negative response for mutagenicity.

Executive summary:

Stepan TAB-2 was tested in the Salmonella and E. coli Mutagenicity Assay using tester strains TA98, TA199, TA 1537, TA1538 and WP2 uvrA in the presence and absence of Aroclor-induced rat liver microsomal enzymes. The assay was performed in two phases using the plate incorporation method. The first phase, the dose range-finding study, was used to establish the dose range for the mutagenicity assay. The second phase, the mutagenicity and confirmatory assays, was used to evaluate the mutagenicity of the test substance.

In the dose range-finding study the maximum dose tested was 10,000 microgram per plate. This dose, limited by the protocol was delivered to the test system as a suspension in acetone. The results of the dose range-finding study indicate that neither appreciable toxicity nor precipitate was observed. Therefore, in compliance with the protocol, the maximum dose that was plated in the mutagenicity assay was 10,000 microgram per plate.

In the mutagenicity assays, precipitate was noted but no appreciable toxicity was observed. In addition, no positive responses were observed.

In conclusion, the results of the Salmonella/Mammalian-Microsome Plate Incorporation Mutagenicity Assay (Ames Test) and Escherichia coli WP2 uvrA Reverse Mutation Assay indicate that under the conditions of this study, test substance Stepan TAB-2, did not cause a positive response with any of the tester strains in the presence and absence of microsomal enzymes prepared from Aroclor-induced rat liver.