Fatty acids, C18-unsatd., compds. with diisopropanolamine N-C12-18-alkyl derivs.

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

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:

2014-11-26 to 2015-05-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Scientifically well performed and well documented Guideline study performed under GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Validity criteria were taken from the literature and DIN-guideline 38415 part 4 since in OECD and EC guideline no validity criteria are described.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

A post-mitochondrial (S9) fraction from livers of male Wistar rats, which received triple treatments of 80 mg/kg body weight Phenobarbital and -Naphtoflavone orally on consecutive days.

Test concentrations with justification for top dose:

Test strain [mg/plate]
1st study (plate incorporation method)
TA 1537 ± S9, TA 98 - S9, TA 100 ± S9, TA 1535 - S9 0.160 – 0.050 – 0.016 – 0.0050 – 0.0016
TA 98 + S9, TA 1535 + S9, E. coli WP2 ± S9 0.500 – 0.160 – 0.050 – 0.016 – 0.0050

2nd study (preincubation method)
TA 98 + S9, E. coli WP2 ± S9 0.500 – 0.23 – 0.050 – 0.023 – 0.0050
TA 1535 + S9 0.500 – 0.23 – 0.050 – 0.023 – 0.0050– 0.0023 – 0.00050 – 0.00023
TA 98 - S9, TA 100 + S9, TA 1537 + S9 0.23 – 0.050 – 0.023 – 0.0050 – 0.0023
TA 100 - S9, TA 1535 - S9, TA 1537 - S9 0.023 – 0.0050 – 0.0023 – 0.00050 – 0.00023

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

double distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

benzo(a)pyrene

other: 4-Nitro-o-phenylen-diamine; Nitroflurantoine; 2-Aminoanthracen

Details on test system and experimental conditions:

Test organism Salmonella typhimurium strains
TA 1537 (not specified),
TA 98 (lot number: 4873D),
TA 100 (lot number: 4846D),
TA 1535 (lot number: 4308D) and
Escherichia coli WP2 (trp, uvrA, pKM101) (lot number: 4854D) with (+) and without (-) metabolic activation system (S9)

Reason for the selection Strains are selected according to the guidelines.
of the test system

Origin TRINOVA BIOCHEM GMBH, Kerkrader Straße 10, D-35394 Gießen

The Salmonella typhimurium lyophilized disc cultures were prepared from master cultures obtained from Dr. Bruce N. Ames, University of California, US.
The Escherichia coli lyophilized disc culture was obtained from the The National Collections of Industrial and Marine Bacteria, Aberdeen, UK.

Storage at test facility Refrigerator, 6 ± 2 °C (TA 98, TA 100, Escherichia coli WP2 (trp, uvrA, pKM101)
Under liquid nitrogen (TA 1537, TA 1535)

Inoculum An overnight culture was prepared. Incubation was performed for 18 ± 2 hours at 37 °C. Titer of the overnight culture must be more than 100000000 cells/mL.

Evaluation criteria:

Since a reduced background lawn is regarded to be a cytotoxic
effect, plates with reduced background lawn normally were not included into evaluation procedures.

Arithmetic mean values and standard deviations were calculated out of colonies per plate of three replicates.

For evaluation of the results the induction rate of the mean
values were calculated:



The test item is to be classified mutagenic if there is a concentration effect relationship over at least three concentrations and/or the induction rate is more than 2.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Validity Criteria

• The genotypes of the tested strains have been confirmed:
- Histidine- and tryptophan auxotrophy, respectively
- Ampicillin resistance
- UV-sensitivity
- growth inhibition with crystal violet (rfa-mutation)

• Titer of the overnight cultures was > 100000000 cells/mL.

• Spontaneous mutation rates (negative controls) met the requirements. For TA 100 (with and without S9 in DMSO and double distilled water, 2nd study) and TA 1535 (without S9 in DMSO, 1st study) the spontaneous mutation rate was higher than the requested range. This deviation was considered to have no impact on quality and integrity of the study.

• The induction rates of the positive controls were > 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Test of the Reference Items

 

Positive controls were tested parallel to the test item.

 

 

Table5: Induction Rates of Reference Items (Positive Controls)           

 

Strain	Reference item	[µg/plate]	S9	Induction Rate*
				1^ststudy	2^ndstudy
TA 1537	ICR 191	2.0	-	> 111	> 51.7
	2-Aminoanthracene	2.0	+	23.2	> 273
TA 98	4-Nitro-o-phenylenediamine	0.5	-	7.0	3.3
	Benzo(a)pyrene	10	+	19.8	6.4
TA 100	Nitrofurantoine	0.2	-	2.7	2.5
	2-Aminoanthracene	2.0	+	10.5	> 4.0
TA 1535	Sodium azide	0.25	-	3.1	4.1
	2-Aminoanthracene	2.0	+	14.9	16.4
E. coliWP2 (uvrA, pKM101)	4-Nitroquinolin-1-oxid	1.0	-	> 6.0	> 4.6
	2-Aminoanthracene	25.0	+	> 3.1	> 4.6

 

Plates with number of colonies higher than 1000 colonies were not counted,

induction rates were calculated with this number and given as > values.

 

*) Induction Rates were calculated in relation to the double distilled water control

Conclusions:

Interpretation of results (migrated information):
negative

In this study Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis, N-C12-18-(even numbered) alkyl derivs. was found to have no mutagenic effects on Salmonella typhimurium strains TA 1537, TA 98, TA 100, TA 1535 and Escherichia coli WP2 (uvrA) with (+) and without (-) the metabolic activation system S9 from male Wistar rats.

Executive summary:

The mutagenic effects of the test item Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis, N-C12-18-(even numbered) alkyl derivs.(batch number: DEG4273438) were determined in a bacterial reverse mutation test according to OECD Guideline No. 471 for Testing of Chemicals (1997) / Directive 2000/32/EC Method B. 13/14 (2000) in two independent studies (plate incorporation method and preincubation method). Test systems were the Salmonella typhimurium strains TA 1537, TA 98, TA 100 and TA 1535 and Escherichia coli  WP2 (uvrA) with (+) and without (-) the metabolic activation system S9 (from male Wistar rats), respectively. Positive and negative controls were included in each study. The test item was dissolved in DMSO and applied once at the start of the exposure with the concentration ranges as given in the Table. Duration of each study was 48 hours. Mutagenic and cytotoxic effects are summarized in the Table.

 

Table:    Mutagenic and Cytotoxic Effects of the Test Item 

 

Strain	S9	Tested Concentration Range [mg/plate]		Lowest Mutagenic Concentration   [mg/plate]	Lowest Cytotoxic Concentration [mg/plate]
		1^ststudy factorÖ10	2^ndstudy factorÖ5		1^ststudy factorÖ10	2^ndstudy factorÖ5
TA 1537	-	0.16 – 0.0016	0.023 – 0.00023	none	0.16	0.023
	+	0.16 – 0.0016	0.23 – 0.0023	none	> 0.16	> 0.23
TA 98	-	0.16 – 0.0016	0.23 – 0.0023	none	> 0.16	0.23
	+	0.5 – 0.005	0.5 – 0.005	none	> 0.5	> 0.5
TA 100	-	0.16 – 0.0016	0.023 – 0.00023	none	0.16	> 0.023
	+	0.16 – 0.0016	0.23 – 0.0023	none	0.16	> 0.23
TA 1535	-	0.16 – 0.006	0.023 – 0.00023	none	> 0.16	0.023
	+	0.5 – 0.005	0.5 – 0.00023	none	> 0.5	0.5
E. coliWP2 (uvrA)	-	0.5 – 0.005	0.5 – 0.005	none	> 0.5	> 0.5
	+	0.5 – 0.005	0.5 – 0.005	none	> 0.5	> 0.5

 

  Result: The test item is regarded to be not mutagneic under this test conditions.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

In this AMES test Fatty acids, C18-unsatd., compd. with 2-propanol, 1,1’-iminobis, N-C12-18-(even numbered) alkyl derivs. was found to have no mutagenic effects on Salmonella typhimurium strains TA 1537, TA 98, TA 100, TA 1535 and Escherichia coli WP2 (uvrA) with (+) and without (-) the metabolic activation system S9 from male Wistar rats.

Justification for selection of genetic toxicity endpoint
Scientifically well performed and well documented Guideline study performed under GLP.

Justification for classification or non-classification

For gentetic toxicity, no classification is warranted for the registration substance according to the OECD GHS criteria.