Isooctadecanoic acid, monoester with propane-1,2-diol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 Jan - 23 Mar 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Amt für Arbeitsschutz - Arbeitnehmerschutz, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1323-39-3/29013-28-3

IUPAC Name:

1323-39-3/29013-28-3

Test material form:

solid

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male rats treated with Aroclor 1254

Test concentrations with justification for top dose:

31.6, 100, 316, 1000, 3160 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol (concentration in vehicle: 10 ng/mL or 20 ng/mL)
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water and DMSO, ethanol was selected as vehicle.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Sodium Azide (SA), 2-Nitrofluorene (2-NF), 9-Aminoacridine (9-AA), Mitomycin C (MC), Benzo(a)pyrene (BaP), 2-Aminoanthracene (2-AA)

Remarks:

-S9: 2-NF (10 µg/pl in DMSO; TA 98); SA (10 µg/pl in water; TA 100 and TA 1535); 9-AA (100 µg/pl in ethanol; TA 1537), MC (10 µg/pl in water; TA 102); +S9: 2-AA (2 µg/pl in DMSO; TA 100 and TA 1535); BaP (10 µg/pl in DMSO; TA 98, TA 102 and TA 1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: pre-incubation

DURATION
- Preincubation period: 20 min (second experiment)
- Exposure duration: 48 - 72 h (first and second experiment)

NUMBER OF REPLICATIONS: triplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants by at least 50%, a clearing or diminution of the background lawn or by the degree of survival of the treated cultures

Evaluation criteria:

A test item is considered to have mutagenic potential, if a significant (p ≤ 0.05) increase of revertant colonies per plate is determined in both experiments (increase factor ≥ 2 for TA 98, TA 100, TA 1535 and TA 1537 or increase factor ≥ 1.5 for TA 102) in at least one strain or a concentration-related increase over the range tested is observed.

Positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on histidine-free agar plates.
Biological relevance of the results should be considered first.

A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Statistics:

Mean values and standard errors were calculated.
Statistical significance was determined via U-test according to MANN and WHITNEY (increase in revertants). In case that a concentration-related increase over the range tested was observed, a Spearman's rank correlation coefficient was applied.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

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:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test item was fully soluble in stock concentrations of 10 and 20 mg/mL. Stock dilutions with higher concentrations (31.6 and 50 mg/mL or 63.2 and 100 mg/mL (corresponding to final concentrations of 3160 or 5000 µg/plate)) were emulsions. Precipitation was noted at 5000 µg/plate in all strains.
- Other: For the positive control substances, no respective solvent control was included. However, due to the range of the mutagenic response, a comparison to the vehicle control ethanol is considered as acceptable.

RANGE-FINDING/SCREENING STUDIES:
A preliminary test was performed in tester strain TA 100 to determine cytotoxcity. Concentrations ranging from 0.316 to 5000 µg/plate were tested in a plate incorporation test without and with metabolic activation. No signs of cytotoxicity were noted in any concentration. Precipitation was observed at 5000 µg/plate. Based on the results of the preliminary study, 5000 µg/plate was chosen as maximum dose for the main study.

COMPARISON WITH HISTORICAL CONTROL DATA:
The mutant frequencies of the solvent controls were within the range of historical control data for any strain. Furthermore, the results of the positive control cultures were within the range of the historical data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
To prevent cytotoxicity of the solvent ethanol in the preincubation test, the volume of the test item and negative control was reduced from the generally employed 100 µL to 50 µL per plate as the preincubation test is more sensitive than the plate incorporation test.

Any other information on results incl. tables

Table 2. Results of the plate incorporation test

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± standard deviation
		Base-pair substitution type			Frameshift type
		TA 100	TA 102	TA 1535	TA 98	TA 1537
–	0 (100 µL/plate)	173.0 ± 17.3±	277.0 ± 21.8	26.3 ± 2.3	29.0 ± 3.6	7.7 ± 2.1
–	31.6	152.3 ± 20.2	265.0 ± 11.4	25.3 ± 1.5	30 ± 7.9	4.3 ± 1.2
–	100	158.0 ± 6.0	267.3 ± 7.6	28.0 ± 3.6	26.7 ± 9.0	4.3 ± 1.2
–	316	134 ± 21.3	268.3 ± 7.8	29.7 ± 6.7	30.0 ± 3.6	4.7 ± 2.1
–	1000	160 ± 8.9	269.3 ± 7.8	37.3 ± 2.9	29.7 ± 0.6	5.0 ± 3.5
–	3160	164.3 ± 2.5	262.0 ± 14.4	28.0 ± 8.9	29.3 ± 1.2	5.3 ± 0.6
–	5000	170p ± 2.0	247.0p ± 2.0	27.7p ± 3.5	33.3p ± 9.0	2.7p ± 0.6
–	Positive controls	SA	MC	SA	2NF	9AA
	Mean No. of colonies/plate ± SD	1023.3 ± 50.0	1119.3 ± 7.0	130.7 ± 17.6	116.7 ± 6.0	61 ± 3.6
+	0 (100 µL/plate)	146.3 ± 15.1	266.7 ± 24.8	26.7 ± 4.0	40.7 ± 6.7	6.7 ± 2.9
+	31.6	121.7 ± 0.6	251.0 ± 1.0	27.7 ± 1.2	36.0 ± 5.2	6.0 ± 1.0
+	100	116.7 ± 7.0	255.3 ± 4.2	32.3 ± 6.4	30.3 ± 0.6	7.3 ± 1.5
+	316	113.7 ± 6.4	282.0 ± 2.6	29.7 ± 3.1	29.0 ± 1.7	5.0 ± 1.0
+	1000	109.3 ± 5.0	221.3 ± 98.2	26.3 ± 1.2	27.0 ± 1.0	4.3 ± 0.6
+	3160	164.0 ± 5.2	272.0 ± 3.6	25.3 ± 2.5	27.7 ± 2.9	4.7 ± 1.5
+	5000	148.0p ± 22.5	270.7p ± 3.1	25.3p ± 1.2	32p ± 2.0	2.3p ± 0.6
+	Positive controls	2AA	BaP	2AA	BaP	BaP
	Mean No. of colonies/plate ± SD	986.3± 2.1	1081.7± 10.5	125.3 ± 18.0	120.7± 3.5	62.7± 0.6

SA = Sodium Azide

MC = Mitomycin C

2NF = 2-Nitrofluorene

9AA = 9 -Aminoacridine

2AA = 2 -Aminoanthracene

BaP = Benzo(a)pyrene

SD = standard deviation

p = precipitate

 

Table 3. Results of the preincubation test

With or without S9-Mix	Test substance concentration (μg/plate)	Mean number of revertant colonies per plate ± standard deviation
		Base-pair substitution type			Frameshift type
		TA 100	TA 102	TA 1535	TA 98	TA 1537
–	0 (50 µL/plate)	147.7± 17.2	263.0 ± 1.0	19.3 ± 3.8	31.0 ± 8.0	6.0 ± 1.0
–	31.6	147.3 ± 6.1	279.7 ± 11.6	20.3 ± 1.2	26.0 ± 2.0	7.3 ± 2.9
–	100	159.3 ± 3.8	280.3 ± 9.0	25.3 ± 4.0	24.7 ± 4.2	8.3 ± 1.2
–	316	137.0 ± 3.0	282.7 ± 8.0	19.7 ± 2.1	26.0 ± 2.0	5.7 ± 1.2
–	1000	137.0 ± 3.6	283.7 ±3.2	19.0 ± 1.0	23.3 ± 1.5	6.7 ± 1.5
–	3160	117.0 ± 114.0	260.0 ± 16.5	23.7 ± 1.5	39.0 ± 3.5	5.7 ± 0.6
–	5000	103.0p ± 2.0	245.3p ± 0.6	8.7p ± 0.6	14.0p ± 2.0	3.3p ± 2.3
–	Positive controls	SA	MC	SA	2NF	9AA
	Mean No. of colonies/plate ± SD	882.7 ± 16.8	1057.7 ± 57.0	147.7 ± 4.0	164.7 ± 8.5	67.3 ± 0.6
+	0 (50 µL/plate)	134.3 ± 4.2	271.3 ± 3.1	20.7 ± 3.8	29.0 ± 4.4	7.3 ± 1.2
+	31.6	128.0 ± 1.7	276.0 ± 12.8	27.0 ± 1.0	32.0 ± 1.0	7.0 ± 2.6
+	100	134.7 ± 8.1	271.7 ± 0.6	26.3 ± 0.6	32.3 ± 0.6	5.3 ± 2.1
+	316	140.7 ± 30.7	268.3 ± 2.1	28.7± 2.1	30.3 ± 2.1	7.7 ± 0.6
+	1000	138.3 ± 1.5	277.0 ± 13.9	26.3 ± 0.6	28.0 ± 1.7	7.7 ± 0.6
+	3160	122.0 ± 9.6	272.3 ± 1.5	27.0 ± 1.0	28.7± 0.63	4.0 ± 1.0
+	5000	102.0p ± 2.6	244.0p ± 1.0	8.0p ± 1.0	13.7p ± 1.2	2.0p ± 0.0
+	Positive controls	2AA	BaP	2AA	BaP	BaP
	Mean No. of colonies/plate ± SD	889.3 ± 12.7	1089.0 ± 14.7	15.03 ± 1.5	168.3 ± 3.2	61.3 ± 10.8

SA = Sodium Azide

MC = Mitomycin C

2NF = 2-Nitrofluorene

9AA = 9 -Aminoacridine

2AA = 2 -Aminoanthracene

BaP = Benzo(a)pyrene

SD = standard deviation

p = precipitate

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative.