Fatty acids, C16-18, reaction products with diethylenetriamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

other: read across from analogue substance

Adequacy of study:

key study

Study period:

28 September 2015 - 27 October 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Amides, from diethylenetriamine and hydrogenated palm oil

EC Number:

810-543-2

Cas Number:

1618093-67-6

Molecular formula:

n/a

IUPAC Name:

Amides, from diethylenetriamine and hydrogenated palm oil

Test material form:

solid: compact

Details on test material:

- Name of test material (as cited in study report): Amidoamine (UVCB)
- Substance type: Amidoamine
- Chemical name: Glycerides, C14-20, reaction products with diethylenetriamine (preliminary naming)/Amides, from diethylenetriamine and hydrogenated palm oil
- CAS 85409-11-6/1618093-67-6
- Physical state: pale yellowish solid at 20 °C
- Batch No.: K8 4309 L481
- Expiry date of batch: 09 March 2017
- Purity: 100 % (UVCB)
- Storage condition of test material: Room temperature, protected from light
- Stability: stable under test conditions

Specific details on test material used for the study:

Test item without emulsifier was investigated.

Amidoamine (UVCB)
Pulcra ID: DE07_2014_012_BEL66 (amidoamine without emulsifier)
Physical state: pale yellowish solid at 20 °C
Batch No.: K8 4309 L481
Expiry date of batch: 09 March 2017
Purity: 100 % (UVCB)
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light

Method

Target gene:

Salmonella typhimurium

Strains / Genotype
TA1537 / his C 3076; rfa-; uvrB-;
TA98 / his D 3052; rfa-; uvrB-;R-factor
TA1535 / his G 46; rfa-; uvrB-
TA100 / his G 46; rfa-; uvrB-;R-factor

Escherichia coli

Strain / Genotype
WP2uvrA / trp-; uvrA-

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 1.5 to 5000 µg/plate.
Six test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.

Vehicle / solvent:

The test item was considered to have formed the best doseable suspension in sterile distilled water (12.5 mg/mL), therefore, this solvent was selected as the vehicle.

Controls

Untreated negative controls:

yes

Remarks:

sterile distilled water

Negative solvent / vehicle controls:

yes

Remarks:

sterile distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene (with metabolic activation), Benzo(a)pyrene (with metabolic activation)

Details on test system and experimental conditions:

The test item was tested using the following method. The maximum concentration was 5000 µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method. All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to colonies spreading and artefacts on the plates, thus distorting the actual plate count.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

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

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

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

Test results 3

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

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

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

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

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

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

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

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

Test results 7

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

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

Test results 8

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

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

Test results 9

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

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

Test results 10

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

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:

Experiment 1 and 2 - plate incorporation test and in the preincubation test.

The S9 Microsomal fraction was prepared in-house from male rats induced with Phenobarbitone/beta-Naphthoflavone at 80/100 mg/kg/day, orally, for 3 days prior to preparation on day 4. The S9 homogenate was produced by homogenizing the liver in a 0.15M KCl solution (1g liver to 3 mL KCl) followed by
centrifugation at 9000 g. The protein content of the resultant supernatant was adjusted to 20 mg/mL. Aliquots of the supernatant were frozen and stored at approximately -196 °C. Prior to use, each batch of S9 was tested for its capability to activate known mutagens in the Ames test.

Remarks on result:

other:

Remarks:

Experiment 1 & 2

Any other information on results incl. tables

Exemplary results of Ames-Test: Plate incorporation - number of revertants (mean) +/- SD for all tested doses with and without metabolic activation.

Experiment 1 – Without Metabolic Activation

S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
	Solvent Control (Water)	119 107 119 (115) 6.9#	11 17 15 (14) 3.1	21 21 21 (21) 0.0	19 12 17 (16) 3.6	7 15 8 (10) 4.4
	1,5 µg	93 103 81 (92) 11.0	16 12 9 (12) 3.5	25 19 32 (25) 6.5	23 19 21 (21) 2.0	15 7 17 (13) 5.3
	5 µg	97 97 114 (103) 9.8	14 10 9 (11) 2.6	27 28 15 (23) 7.2	15 12 19 (15) 3.5	12 7 16 (12) 4.5
	15 µg	94 126 110 (110) 16.0	14 12 13 (13) 1.0	19 24 32 (25) 6.6	28 11 11 (17) 9.8	9 15 7 (10) 4.2
	50 µg	117 129 114 (120) 7.9	9 9 10 (9) 0.6	17 21 19 (19) 2.0	17 20 23 (20) 3.0	11 11 11 (11) 0.0
	150 µg	94 112 118 (108) 12.5	14 11 10 (12) 2.1	16 21 24 (20) 4.0	13 12 27 (17) 8.4	7 9 12 (9) 2.5
	500 µg	103 95 107 (102) 6.1	8 10 16 (11) 4.2	32 36 17 (28) 10.0	21 25 12 (19) 6.7	9 12 15 (12) 3.0
	1500 µg	109 91 110 (103) 10.7	6 12 10 (9) 3.1	23 13 25 (20) 6.4	11 8 24 (14) 8.5	5 9 12 (9) 3.5
	5000 µg	94 P 115 P 124 P (111) 15.4	9 P 12 P 5 P (9) 3.5	29 P 36 P 26 P (30) 5.1	13 P 13 P 23 P (16) 5.8	8 P 4 P 11 P (8) 3.5
Positive controls S9-Mix (-)	Name	ENNG	ENNG	ENNG	4NQO	9AA
	Dose Level	3 µg	5 µg	2 µg	0.2 µg	80 µg
	No. of Revertants	386 392 444 (407) 31.9	298 358 372 (343) 39.3	579 521 476 (525) 51.6	192 210 178 (193) 16.0	964 869 671 (835) 149.5

†               Experimental procedure repeated at a later date due to poor colony growth in the original test

ENNG      N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO      4-Nitroquinoline-1-oxide

9AA         9-Aminoacridine

P               Test item precipitate

#               Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains			Frameshift strains
		TA100	TA1535	WP2uvrA	TA98	TA1537
	Solvent Control (Water)	110 115 124 (116) 7.1#	12 11 8 (10) 2.1	20 28 29 (26) 4.9	16 20 16 (17) 2.3	8 9 13 (10) 2.6
	1,5 µg	117 125 119 (120) 4.2	8 12 10 (10) 2.0	23 32 25 (27) 4.7	15 15 23 (18) 4.6	7 17 12 (12) 5.0
	5 µg	118 106 126 (117) 10.1	13 9 5 (9) 4.0	17 36 25 (26) 9.5	11 29 19 (20) 9.0	15 7 15 (12) 4.6
	15 µg	114 129 121 (121) 7.5	17 9 9 (12) 4.6	29 29 16 (25) 7.5	16 28 29 (24) 7.2	8 11 7 (9) 2.1
	50 µg	82 122 119 (108) 22.3	8 8 8 (8) 0.0	31 23 29 (28) 4.2	23 19 15 (19) 4.0	9 13 13 (12) 2.3
	150 µg	91 97 94 (94) 3.0	11 11 12 (11) 0.6	28 20 23 (24) 4.0	28 27 19 (25) 4.9	17 19 17 (18) 1.2
	500 µg	106 106 99 (104) 4.0	8 7 12 (9) 2.6	33 39 27 (33) 6.0	20 21 25 (22) 2.6	13 8 7 (9) 3.2
	1500 µg	117 97 90 (101) 14.0	10 11 7 (9) 2.1	27 27 17 (24) 5.8	28 17 20 (22) 5.7	5 13 11 (10) 4.2
	5000 µg	102 P 98 P 97 P (99) 2.6	11 P 7 P 5 P (8) 3.1	25 P 28 P 20 P (24) 4.0	16 P 32 P 33 P (27) 9.5	16 P 15 P 9 P (13) 3.8
Positive controls S9-Mix (+)	Name	2AA	2AA	2AA	BP	2AA
	Dose Level	1 µg	2 µg	10 µg	5 µg	2 µg
	No. of Revertants	719 647 540 (635) 90.1	243 221 215 (226) 14.7	372 351 405 (376) 27.2	195 183 207 (195) 12.0	342 376 341 (353) 19.9

†            Experimental procedure repeated at a later date due to poor colony growth in the original test

BP         Benzo(a)pyrene

2AA      2-Aminoanthracene

P            Test item precipitate

#            Standard deviation

Applicant's summary and conclusion

Conclusions:

The test item was considered to be non-mutagenic under the conditions of this test.

Executive summary:

In a key Ames test performed with registered substance, the test item was examined in the four Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and one Escherichia coli strain in two independent experiments, each carried out without and with metabolic activation (P Thompson, 2016). The first experiment was carried out as a plate incorporation test and the second as a preincubation test. The test item was suspended in sterile distilled water. Sterile distilled water was used as vehicle control. In a preliminary test, no cytotoxicity was noted at concentrations up to 5000 µg test item/plate, hence 5000 µg test item/plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test. In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, no increase in revertant colony numbers as compared with control counts was observed up to a concentration of 5000 µg test item/plate, in any of the 5 test strains. A test item precipitate (powdery in appearance) was observed at 5000 µg/plate in the first mutation test (plate incorporation method) and initially from 500 µg/plate in the second mutation test (pre-incubation method), this observation did not prevent the scoring of revertant colonies. In conclusion, negative results were obtained for both bacterial mutagenicity studies tested both with and wihtout metbabolic activation.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method).