Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is no experimental data regarding the mutagenic potential of the target substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1). However, literature data is available forin vitro assays which have been conductedon the constituents this salt, Octanoic acid (CAS 124-07-2) and Triethanolamine (CAS 102-71-6). As the Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is expected to freely dissociate in water, to yield free Octanoic acid and Triethanolamine, the toxicological properties of Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) will be closely related to its constituents. The use of the available mutagenicity data of these constituents to address thein vitro mutagenic potential of the target substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was therefore deemed scientifically appropriate and justified.

 

The hypothesis that Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) will readily dissociate is supported by the available experimental data. The high water solubility of this substance suggests that the ion pairs freely dissociate and the corrosivity result obtained after > 3 mins to < 1 hour in the OECD 431 guideline study (XCELLR8, 2019) suggests that free acid is available.

 

Thein vitromutagenic potential of 270 chemicals, including Triethanolamine, was investigated by Mortelmans K.et al (1986) using the preincubation assay as part of a screening programme for the NTP, in a study similar to OECD guideline 471. Under the conditions of the study, Triethanolamine was found to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

 

In the same report, as part of the same NTP screening programme, thein vitro mutagenicity result for the structurally analogous substance TEA Stearate (Stearic acid, compound with 2,2',2''-nitrilotriethanol (1:1), CAS 4568-28-9) is also reported. The result was negative, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

 

In similar study reported by Zeiger, E.et al (1988), the in vitro mutagenic potential of Octanoic acid (CAS 124-07-2) was investigated as part of the NTP screening programme. Under the conditions of this study Octanoic acid was also found to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97, TA98 and TA 100.

 

The ability of Triethanolamine to induce chromosome aberrations and sister chromatid exchanges in Chinese Hamster Ovary Cells has also been investigated and reported by Gallowayet al (1987). The result of this in vitro assay was negative, both in the presence and absence of metabolic activation.

 

As no evidence of genotoxic activity was observed in the experimental in vitro assays conducted and reported in literature for Octanoic acid (CAS 124-07-2), Triethanolamine (CAS 102-71-6) and TEA Stearate (Stearic acid, compound with 2,2',2''-nitrilotriethanol (1:1), CAS 4568-28-9), the substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was determined to be non-mutagenic by application of read-across.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

As octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is expected to readily dissociate into Triethanolamine and Octanoic acid, it is considered appropriate to read across from Octanoic Acid and Triethanolamine. Octanoic acid data is from a peer reviewed scientific journal.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

strain with AT base pair as primary reversion site missing

Principles of method if other than guideline:

Test protocol is decribed by Haworth et al., 1983. No strains with AT base pair at the primary reversion site were tested.

[Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity results for 250 chemicals. Environ Mutagen 5(Suppl 1):3-142.]

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Publication states chemical source as Aldrich Chemical, with a label purity of 99.5% and an analyzed purity of >99%.

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster metabolic activation

Test concentrations with justification for top dose:

0, 10, 33, 100, 333, 1000, 3333 µg/plate
All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the system developed by Waleh et al. [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-o-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Strains
Salmonella typhimunum strains TA97, TA98, TA100, TA1535, and TA1537 were obtained from Dr. Bruce Ames (University of California, Berkeley) and were stored as recommended [Maron and Ames, 1983]. Cultures were grown overnight with shaking at 37°C in Oxoid No. 2 broth, and their phenotypes were analyzed prior to their use for mutagenicity assays.

Preparation of S-9 Fraction
The S-9 (9,000g supernatant) fractions of Aroclor 1254-induced, male Sprague- Dawley rat and male Syrian hamster livers were prepared as described previously [Haworth et al, 1983]. The S-9 mixes were prepared immediately prior to use and contained either 10% or 30% S-9; occasionally, other levels were used. The specific levels used are indicated in Appendix 2. All chemicals were tested in the absence of metabolic activation and with rat and hamster S-9 fractions.

Preincubation Assay
The preincubation assay was performed as described previously [Haworth et al., 1983], with some differences, as described below. The test chemical (0.05 ml), Salmonella culture (0.10 ml), and S-9 mix or buffer (0.50 ml) were incubated at 37°C, without shaking, for 20 min. Chemicals known or suspected to be volatile were incubated in capped tubes. The top agar was added and the contents of the tubes were mixed and poured onto the surface of petri dishes containing Vogel-Bonner medium [Vogel and Bonner, 1956]. The histidine-independent (his') colonies arising on these plates were counted following two days incubation at 37°C. Plates were machine counted (New Brunswick, Edison, NJ; Artek, Farmingdale, NY) unless precipitate was present which interfered with the count, or the colour of the test chemical on the plate reduced the contrast between the colonies and the background agar. At the discretion of the investigators, plates with low numbers of colonies were counted by hand.
Variations in the protocol among the tested chemicals reflect the evolution of the protocol originally described by Haworth et al. [1983]. Four protocol variations are evident from the data in Appendix 2. 1) Testing in strains TA97, TA98, TA100, and TA1535, with some additional testing in strain TA1537; 10% S-9 was used. 2) The first test of a chemical was without activation and with 10% S-9 in the S-9 mix. If a positive result was obtained the test was repeated. If the tests were negative they were repeated without S-9 and with 30% S-9. 3) The order of use of 10% and 30% S-9 was reversed. 4) Initial testing was in strains TA98 and TA100 without activation and with 30% rat and hamster S-9s. If a positive result was obtained in one of these two strains it was repeated and the other strains were not used. If the tests were negative, the other strains were used with 30% and 10% S-9. A chemical was not designated nonmutagenic unless it had been tested in strains TA98, TA100, TA1535, and TA97 and/or TA1537, without activation and with 10% and 30% rat and hamster S9. Occasionally, 5% S-9 was also used in all protocol variations. All chemicals were tested initially in a toxicity assay to determine the appropriate dose range for the mutagenicity assay. The toxicity assay was performed using TA100 or the sytem developed by Waleh et al. [1982]. Toxic concentrations were those that produced a decrease in the number of his+ colonies, or a clearing in the density of the background lawn, or both.
Each chemical was tested initially at half-log dose intervals up to a dose that elicited toxicity, or to a dose immediately below one which was toxic in the preliminary toxicity test. Subsequent trials occasionally used narrower dose increments and may not have included doses in the toxic range. Chemicals that were not toxic were tested, with few exceptions, to a maximum dose of 10 mg/plate.
Chemicals that were poorly soluble were tested up to doses defined by their solubilities. At least five doses of each chemical were tested in triplicate. Experiments were repeated at least one week following the initial trial. A maximum of 0.05 ml solvent was added to each plate. Concurrent solvent and positive controls were run with each trial. The positive controls in the absence of metabolic activation were sodium azide (TA1535 andTA100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control for metabolic activation with all strains was 2-aminoanthracene.

Evaluation criteria:

The data were evaluated as described previously [Zeiger et al., 1987]. Evaluations were made at both the individual trial and overall chemical levels. Individual trials were judged mutagenic (+), weakly mutagenic (+ W), questionable (?), or non-mutagenic (-), depending on the magnitude of the increase of his+ revertants, and the shape of the dose-response. A trial was considered questionable (?) if the dose-response was judged insufficiently high to support a call of “ +W,” if only a single dose was elevated over the control, or if the increase seen was not dose related. The distinctions between a questionable mutagenic response and a non-mutagenic or weak mutagenic response and between a weak mutagenic response and mutagenic response are highly subjective. It was not necessary for a response to reach two-fold over background for a chemical to be judged mutagenic. A chemical was judged mutagenic (+) or weakly mutagenic (+ W) if it produced a reproducible dose-related response over the solvent control in replicate trials. A chemical was judged questionable (?) if the results of individual trials were not reproducible, if increases in his+ revertants did not meet the criteria for a “+W” response, or if only single doses produced increases in his+ revertants in repeat trials. Chemicals were judged non-mutagenic (-) if they did not meet the criteria for a mutagenic or questionable response. The chemicals were decoded by the chemical repository only after a determination had been made regarding their mutagenicity or non-mutagenicity.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this publication, Octanoic acid (CAS 124-07-2) is reported to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA1537, TA97, TA98 and TA100.

Executive summary:

In a publication entitled "Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals " (Environ Molec Mutagen, 11, Suppl.12 ,pg 1 -158, (1988);.Zeiger, E. et al), the in vitro mutagenic potential of 300 chemicals, including Octanoic acid (CAS 124 -07 -2), was investigated.

 

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimischet al. (1997) as study is well documented and the method described in the literature is similar to the guideline study with the exception that no strains which have an AT base pair at the primary revision site were tested.

Mutagenicity was investigated using the preincubation assay as part of a screening programme for the NTP. Under the conditions of the study Octanoic acid was found to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97, TA98 and TA 100.

Reference 2

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

As octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is expected to readily dissociate into Triethanolamine and Octanoic acid, it is considered appropriate to read across from Octanoic Acid and Triethanolamine. Triethanolamine data is from a peer reviewed scientific journal.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Principles of method if other than guideline:

Method is desrcibed in detail in Galloway SM, Bloom AD, Resnick M, Margolin BH, Nakamura F, Archer P, Zeiger E (1985): Development of a standard protocol for in vitro cytogenetic testing with Chinese hamster ovary cells: Comparison of results for 22 compounds in two laboratories. Environ Mutagen 7: 1–51.

GLP compliance:

not specified

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured in Mc-Coy's 5a medium with 10% fetal calf serum, L-glutamine, and antibiotics

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

100 - 2520 µg/ml (without S9)
330 - 10100 µg/ml (with S9)
In the first SCE test with each chemical, cells were exposed to a range of doses spanning four to five orders of magnitude, in half-log increments, up to a maximum dose of 5-10 mg/ml or to the limits of solubility in culture medium. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

Vehicle / solvent:

Water, dimethyl suifoxide (DMSO), ethanol, or acetone, in that order of preference.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

5-Bromodeoxyuridine (BrdUrd; 10 μM) was added 2 hr after addition of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were approximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1 μg/ml) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of
mitotic cells were noted. Cells were collected by mitotic shake-off at doses up to the maximum considered likely to yield sufficient metaphase cells for analysis; supernatant medium was returned to appropriate flasks so that subsequent harvests could be made from the same cultures if necessary. Because all mitotic cells were removed in the initial harvest, cells collected during subsequent harvests had come into mitosis during the period between harvests and thus had been exposed to colcemid for an average of 4 hr. After 1-3 min treatment with hypotonic solution (75 mM KCl), cells were fixed in 3: 1 methan1:glacial acetic acid (V/V). For a preliminary assessment of cell cycle delay, test slides were prepared from cells treated at the highest dose levels to see if later harvests were necessary. These test slides were stained with “dilute” Hoeschst 33258 (0.5 μg/ml in Sorensen’s buffer, pH 6.8) and examined by fluorescence microscopy to assess cell cycle kinetics. In control cultures, almost all cells completed two cycles in BrdUrd (M2 cells) in 25-26 hr, whereas, in treated cultures, cell cycle delay was common. In cases of severe delay, additional harvests were made from the same cultures at a later time to obtain sufficient second metaphase (M2) cells for SCE analysis. For scoring SCEs, slides were stained by a modification of the method of Perry and Wolff [1974] as adapted by Goto et al [1978]. After staining for 10 min in “concentrated” Hoechst 33258 (5 μg/ml in pH 6.8 buffer) and exposure to “black light” at 55 to 60°C for about 5 min, slides were stained in Giemsa. All slides were coded, and 50 cells per dose were scored from the three highest doses at which sufficient M2 cells were available, from a solvent control, and from a “weak positive” control (in later experiments) treated with a low dose of mitomycin C (without S9) or cyclophosphamide (with S9). This weak-positive control was designed to give a small (20-40%) increase in SCEs and was included to assess the ability of the system to detect small increases in SCEs [Margolin and Resnick, 1985]. After the introduction of the weak-positive control into the protocol, only five to ten cells were scored from “strong-positive” controls (higher doses of mitomycin C or cyclophosphamide) that were not coded. When cell cycle delay was noted, cell kinetics were recorded, at LBI, by classifying each of 100 metaphases as M1, M1+, or M2, ie, having completed one (Ml), two (M2), or between one and two (MI +) cell cycles in BrdUrd.

Evaluation criteria:

In this publication, a linear regression test (trend test) of SCEs per chromosome vs the log of the dose was used [Armitage, 1955]. For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant [Galloway et al, 1985].

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Conclusions:

In this publication, Triethanolamine is reported as negative for the ability to induce chromosome aberrations and sister chromatid exchanges in Chinese hamster ovary cells with and without metabolic activation.

Executive summary:

In a publication entitled "Chromosome Aberrations and Sister Chromatid Exchanges in Chinese Hamster Ovary Cells: Evaluations of 108 Chemicals " (Environ.Molec.Mutag, Vol 10, Suppl 10, pg 1-175 (1987); Galloway et al), the in vitro potential of 108 chemicals, including Triethanolamine (CAS 102 -71 -6), to induce chromosome aberrations and sister chromatid exchanges was investigated.

 

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimisch et al.(1997) as study is well documented and the method described in the literature is similar to the guideline study.

 

Under the conditions of this study, the result was negative. Triethanolamine was not found to cause the induction of chromosome aberrations and sister chromatid exchanges, both in the presence and absence of metabolic activation. The results were negative even up to doses that induced cell cycle delay (SCE test without S9) and toxicity (aberrations with and without S9).

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

As octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is expected to readily dissociate into Triethanolamine and Octanoic acid, it is considered appropriate to read across from Octanoic Acid and Triethanolamine. Triethanolamine data is from a peer reviewed scientific journal.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Four Bacterial strains were used

Principles of method if other than guideline:

The approach and rationale for the testing have been described in detail by Zeiger and Drake, 1980; Haworth et al, 1983.

Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5(Suppl. 1):3-142.
Zeiger E, Drake JW (1980): An environmental mutagenesis test development programme. In Montesano R, Bartsch H, Tomatis L (eds): "Molecular and Cellular Aspects of Carcinogen Screening Tests." Lyon: IARC Scientific Publications No. 27, pp 303-313.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Treithanolamine

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster metabolic activation

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333 μg/ plate

Vehicle / solvent:

Distilled Water

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-O-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Strains
Salmonella strains TA1535, TA1537, TA97, TA98, and TA100 were obtained by the lab from Dr. Bruce Ames (University of California, Berkeley), and were stored according to the protocol described in the supplement to the methods paper [Ames et al, 1975] which was supplied with the strains. Cultures were kept in a -70°C freezer. For overnight inoculation, CWR transferred a loopful of cells, maintained on Columbia agar slants at 4°C, into Columbia broth. All bacterial cultures were grown overnight for 12-15 hr at 37°C on a shaker, and their phenotypes were analyzed as recommended by Ames et al [1975].

Preparation of S-9 Fraction
Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the preparation of the liver fractions. Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to cervical dislocation. The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KCl per gm of wet tissue) in a Potter-Elvehjem apparatus with a Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4 °C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen. The S-9 mix was prepared immediately prior to each assay and consisted of the following, per mL: S-9 fraction, 0.10 ml; 0.04 M MgC12, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M β3-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 ml; 0.05 M glucose-6-phosphate, 0.10 ml; 1.0 M NaH2P04, (pH 7.4), 0.10 ml; and distilled water, 0.56 ml. Other levels of S-9 in the S-9 mix were used for some Aliquots.

Preincubation Assay
All chemicals were assayed for mutagenicity in the preincubation assay [Haworth et al, 1983]. To each of 13 x 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.0 ml of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1956] in Fisher Scientific plates. When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. All assays were repeated (as described above) no less than 1 wk after completion of the initial test.

Evaluation criteria:

The criteria used for data evaluation were the same as those described previously [Haworth et al, 1983], and are summarized as follows: 1) mutagenic response: adose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) non-rnutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, non-rnutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.).

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this publication, Triethanolamine is reported to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

Executive summary:

In a publication entitled "Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals " (Environ. Mutagenesis, 8, Suppl.7 ,pg 1-119, (1986);.Mortelmans K. et al), the in vitro mutagenic potential of 270 chemicals, including Triethanolamine, was investigated.

 

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimischet al. (1997) as study is well documented and the method described in the literature is similar to the guideline study with the exception that no strains which have an AT base pair at the primary revision site were tested.

 

Mutagenicity was investigated using the preincubation assay as part of a screening programme for the NTP. Under the conditions of the study Triethanolamine was found to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed scientific journal. In vitro mutagenicity result for the structurally analogous substance TEA Stearate (Stearic acid, compound with 2,2',2''-nitrilotriethanol (1:1), CAS 4568-28-9).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Four Bacterial strains were used

Principles of method if other than guideline:

The approach and rationale for the testing have been described in detail by Zeiger and Drake, 1980; Haworth et al, 1983.

Haworth S, Lawlor T, Mortelmans K, Speck W, Zeiger E (1983): Salmonella mutagenicity test results for 250 chemicals. Environ Mutagen 5(Suppl. 1):3-142.
Zeiger E, Drake JW (1980): An environmental mutagenesis test development programme. In Montesano R, Bartsch H, Tomatis L (eds): "Molecular and Cellular Aspects of Carcinogen Screening Tests." Lyon: IARC Scientific Publications No. 27, pp 303-313.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Treithanolamine Stearate

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster metabolic activation

Test concentrations with justification for top dose:

0, 33, 100, 333, 1000, 3333, 10000 μg/ plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-O-phenylenediamine; 2-aminoanthracene

Details on test system and experimental conditions:

Bacterial Strains
Salmonella strains TA1535, TA1537, TA97, TA98, and TA100 were obtained by the lab from Dr. Bruce Ames (University of California, Berkeley), and were stored according to the protocol described in the supplement to the methods paper [Ames et al, 1975] which was supplied with the strains. Cultures were kept in a -70°C freezer. For overnight inoculation, CWR transferred a loopful of cells, maintained on Columbia agar slants at 4°C, into Columbia broth. All bacterial cultures were grown overnight for 12-15 hr at 37°C on a shaker, and their phenotypes were analyzed as recommended by Ames et al [1975].

Preparation of S-9 Fraction
Male Sprague-Dawley rats and male Syrian hamsters were routinely used for the preparation of the liver fractions. Aroclor 1254 (200 mg/ml in corn oil) was administered ip at 500 mg/kg 5 days prior to cervical dislocation. The animals were deprived of food 12-24 hr immediately preceding death; otherwise food and water were provided ad libitum. The livers were removed aseptically, washed in ice-cold 0.15 M KCl, and minced and homogenized (3 ml of 0.15 M KCl per gm of wet tissue) in a Potter-Elvehjem apparatus with a Teflon pestle. CWR initially used a Waring blender, but switched to a Potter-Elvehjem apparatus. The S-9 fraction was obtained by centrifugation of the liver homogenate for 10 min at 9,000 g at 4 °C. The S-9 fraction was dispensed into freezing ampules and stored in a -70°C freezer, or in liquid nitrogen. The S-9 mix was prepared immediately prior to each assay and consisted of the following, per mL: S-9 fraction, 0.10 ml; 0.04 M MgC12, 0.02 ml; 1.65 M KCl, 0.02 ml; 0.04 M β3-nicotinamide adenine dinucleotide phosphate (NADP), 0.10 ml; 0.05 M glucose-6-phosphate, 0.10 ml; 1.0 M NaH2P04, (pH 7.4), 0.10 ml; and distilled water, 0.56 ml. Other levels of S-9 in the S-9 mix were used for some Aliquots.

Preincubation Assay
All chemicals were assayed for mutagenicity in the preincubation assay [Haworth et al, 1983]. To each of 13 x 100-mm test tubes maintained at 37°C were added in the following order: 0.5 ml of S-9 mix or 0.1 M PO4 buffer (pH 7.4), 0.05 ml of the overnight culture, and 0.05 ml of solvent or chemical dilution. The mixture was mixed and allowed to incubate without shaking at 37°C for 20 min, at which time 2.0 ml of molten (45°C) top agar supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin were added. The contents of the tubes were mixed and poured onto 25 ml of minimal glucose bottom agar [Vogel and Bonner, 1956] in Fisher Scientific plates. When the top agar had solidified, the plates were inverted and incubated at 37°C for 48 hr. Concurrent solvent and positive controls were tested with and without the metabolic activation systems. At least five dose levels of the chemicals were tested, with three plates per dose level. All assays were repeated (as described above) no less than 1 wk after completion of the initial test.

Evaluation criteria:

The criteria used for data evaluation were the same as those described previously [Haworth et al, 1983], and are summarized as follows: 1) mutagenic response: adose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold; 2) non-rnutagenic response: when no increase in the number of revertants was elicited by the chemical; 3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity. The initial determination of mutagenic, non-rnutagenic, or equivocal was made by the testing laboratory; the final determination was made by the project officer (E.Z.).

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this publication, Triethanolamine stearate is reported to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

Executive summary:

In a publication entitled "Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals " (Environ. Mutagenesis, 8, Suppl.7 ,pg 1-119, (1986);.Mortelmans K. et al), the in vitro mutagenic potential of 270 chemicals, including Triethanolamine Stearate, was investigated.

 

The study was assigned a reliability score of 2 in accordance with the criteria for assessing data quality set forth by Klimischet al. (1997) as study is well documented and the method described in the literature is similar to the guideline study with the exception that no strains which have an AT base pair at the primary revision site were tested.

 

Mutagenicity was investigated using the preincubation assay as part of a screening programme for the NTP. Under the conditions of the study Triethanolamine stearate was found to be non-mutagenic, with and without metabolic activation, in Salmonella typhimurium strains TA1535, TA 1537, TA 97 and TA 100.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro mutagenic potential of the target substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was determined using data from the constituents this salt, Octanoic acid (CAS 124-07-2) and Triethanolamine (CAS 102-71-6). As Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) is expected to freely dissociate in water, the use of the available mutagenicity data of these constituents to address the in vitro mutagenic potential of the target substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) was deemed scientifically appropriate and justified.

Justification for classification or non-classification

There was no evidence of genotoxic activity in the experimental in vitro assays conducted and reported in literature for Octanoic acid (CAS 124-07-2), Triethanolamine (CAS 102-71-6) and TEA Stearate (Stearic acid, compound with 2,2',2''-nitrilotriethanol (1:1), CAS 4568-28-9). Therefore, the substance Octanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1), CAS 22919 -56 -8 was determined to be non-mutagenic, by application of read-across.