Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

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:

Study initiation: 08/01/1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented GLP study performed according to OECD Guideline 471 and EPA OTS 798.5265.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

EC Number:

272-789-1

EC Name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

Cas Number:

68911-83-1

Molecular formula:

C40H76N2O

IUPAC Name:

Fatty acids, tall-oil, reaction products with formaldehyde and (Z)-N-9-octadecenyl-1,3-propanediamine

Details on test material:

- Name of test material (as cited in study report): CI-46C
- Substance type: Clear viscous dark brown liquid
- Physical state: Liquid
- Lot/batch No.: 7783-71395
- Storage condition of test material: Room temperature, protected from exposure to light

Method

Target gene:

Tester strains TA98 and TA1535 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to base-pair mutations, rather than frameshift mutations (Green, MHL, and Muriel WJ (1976). Mutagen testing using trp+ reversion in Escherichia coli. Mutation Research 38: 3-32.

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain 3

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Assay: 6.7, 10, 33, 67, 100, 333, 667, 1000, 3333, 5000 µg/plate
Mutagenicity Assay: 10, 33, 100, 333, 1000, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (100%)
- Justification for choice of solvent/vehicle: Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. THe test article was soluble in ethanol at approximately 500 mg/mL, the maximum concentration tested.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (1.0 µg/plate for all Salmonella strains) (10 µg/plate for WP2 uvrA)

Remarks:

With metabolic activation

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sterigmatocystin (10 µg/plate) for WP2 uvrA

Remarks:

With metabolic activation

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation

Migrated to IUCLID6: 1.0 µg/plate for TA98

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation

Migrated to IUCLID6: 1.0 µg/plate for TA100, TA1535

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation

Migrated to IUCLID6: 75 µg/plate for TA1537

Controls 6

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation

Migrated to IUCLID6: 1000 µg/plate for both E. coli strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation
The test system was exposed to the test article via the plate incorporation methodology originally described by Ames et al. (1975) and updated by Maron and Ames (1983). In the conformatory assay, the test system was exposed to the test article via the preincubation methodology described by Yahagi et al. (1977).
Ames BN, McCann J and Yamasaki E (1975). Methods for Detecting Carcinogens and Mutagens with the Salmonella/Mammalian Microsome Mutagenicity Test, Mutation Research, 31: 347-364
Maron DM and Ames BN (1983). Revised Methods for the Salmonella Mutagenicity Test, Mutation Research, 113: 173-215
Yahagi M, Nagao Y, Seino T, Sugimura T and Okada M (1977). Mutagenicities of N-nitrosamines on Salmonella. Mutation Research 48: 121-130.

DURATION
- Exposure duration: The test article dilutions were prepared immediately before use. In the plate incorporation method, one-half (0.5) milliliter of S9 or Sham mix, 100 µL of tester strain and 50 or 25 µL of vehicle or test article were added to 2.0 mL of molten selective top agar at 45+/- 2°C. After vortexing, the mixture was overlaid onto the surface of 25 mL of minimal bottom agar. In the preincubation method, one-half (0.5) milliliter of S9 or sham mix, 100 µL of tester strain and 25 µL of vehicle or test article were added to 13 x 100 mm glass culture tubes pre-heated to 37 +/- 2°C. After vortexing, these mixtures were incubated without shaking for 60 +/- 2 minutes at tube and the mixture was vortexed and overlaid onto the surface of 25 mL of minimal bottom agar. When plating the positive controls, the test article aliquot was replaced by a 50 µL aliquot of appropriate positive control. After the overlay had solidified, the plates were inverted and incubated for approximately 48 to 72 hours at 37 +/- 2°C. Plates that were not counted immediately following the 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 article toxicity and precipitate by using a dissecting microscope. Toxicity and degree of precipitation were scored relative to the vehicle control plate.
Background bacterial evaluation code:
1 = normal
2 = slightly reduced
3 = Moderately reduced
4 = Extremely reduced
5 = Absent
6 = Oscured by precipitate
SP = Slight precipitate
MP = Moderate precipitate
HP = Heavy precipitate
Revertant colonies for a given tester strain and activation condition were counted either entirely by automated colony counter or entirely by hand unless the assay was the preliminary toxicity assay or the plate exhibited toxicity. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

NUMBER OF REPLICATIONS: All dose levels of test article, vehicle controls and positive controls were plated in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth: A minimum of three non-toxic dose levels will be required to evaluate assay data. A dose level is considered toxic if it causes a > 50% reduction in the mean number of revertants per plate relative to the mean vehicle control value (this reduction must be accompanied by an abrupt dose-dependent drop in the revertant count) or a reduction in the background lawn. In the event that fewer than three non-toxic dose levels are achieved, the affected portion of the assay will be repeated with an appropriate change in dose levels.

Evaluation criteria:

For the test article to be evaluated positive, it must cause a dose-dependent increase in the mean revertants per plate of at least one tester strain with a minimum of two increasing concentrations of test article. Data sets for strains TA 1535 and TA 1537 were 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 WP2 uvrA and WP2 were 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:

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

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was generally observed at 5000 µg per plate with the plate incorporation method and from 33 to 5000 µg per plate with Samonella with the preincubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was generally observed at 5000 µg per plate with the plate incorporation method and from 33 to 5000 µg per plate with Samonella with the preincubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Toxicity was generally observed at 5000 µg per plate with the plate incorporation method and from 33 to 5000 µg per plate with Samonella with the preincubation method.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility test: Ethanol was selected as the solvent of choice based on solubility of the test article and compatibility with the target cells. The test article was soluble in ethanol at approximately 500 mg/mL, the maximum concentration tested.
- Precipitation: Generally, precipitate was observed at > or = 333 and > or = 1000 µg per plate with the plate incorporation and preincubation methods, respectively.

RANGE-FINDING/SCREENING STUDIES:
Preliminary toxicity assay:
In the preliminary toxicity assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot.
Generally, precipitate was observed at > or = 333 and > or = 1000 µg per plate with the plate incorporation and preincubation methods, respectively. Toxicity was generally observed at 5000 µg per plate with the plate incorporation method and from 100 to 5000 µg per plate with Salmonella with the preincubation method.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxicity was generally observed at 5000 µg per plate with the plate incorporation method and from 33 to 5000 µg per plate with Salmonella with the preincubation method.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Mutagenicity assay:

In Experiment B1, the initial mutagenicity assay, no positive responses were observed with any of the tester strains in the presence and absence of S9 activation.

In Experiment B2, the confirmatory assay, no positive responses were observed with tester strains TA98, TA100, TA1535, TA1537 and WP2 uvrA (pKM101) in the presence of S9 activation and with the tester strains TA1535, TA1537 and Wp2 (pKM101) in the absence of S9 activation. Due to excessive toxicity, tester strain WP2 uvrA (pKM101) in the presence of S9 activation were not evaluated but were retested in Experiment B3. To clarify the response, tester strain TA100 in the presence of S9 activation was retested in Experiment B3.

In experiment B3, no positive responses were observed with tester strain WP2 uvrA (pKM101) in the presence of S9 activation and tester strains TA98, TA100 and WP2 uvrA (pKM101) in the absence of S9 activation.

All criteria for a valid study were met as described in the protocol.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The results of the Salmonella/Escherichia coli Mutagenicity Assay indicate that under the conditions of this study, the test substance did not cause a positive response with any of the tester strains in the presence and absence of Aroclor-induced rat liver S9.