Isooctadecanoic acid, mono- and diesters with glycerol

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

18 Oct - 29 Oct 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

736150-63-3

EC Number:

616-005-1

Cas Number:

736150-63-3

IUPAC Name:

736150-63-3

Details on test material:

- Name of test material (as cited in study report): only trade name given
- Chemical name: acetic acid esters of monoglyceride of fully hydrogenated castor oil
- Substance type: clear liquid
- Analytical purity: no data
- Lot/batch No.: 10102
- Expiration date of the lot/batch: 26-04-2002
- Item No.: 175540
- Storage condition of test material: at room temperature, protected from light

Method

Target gene:

his operon

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
Liquid growth medium (broth): Oxoid Nutrient Broth No. 2 (2.5% (w/v) in distilled water
Selective agar plates: Vogel-Bonner medium composed of bacto agar (1.5%), D-glucose (2%), MgSO4 x 7H20 (0.02%), citric acid (0.2%), K2HPO4 (1%), NaNH4HPO4 x 4 H2O (0.35%) in distilled water (percentages correspond to w/v)
The components were autoclaved separately and mixed afterwards.
Top-agar: bacto agar (0.6%), NaCl (0.5%) in distilled water (percentages correspond to w/v), supplemented with 0.05 mM histidine and 0.05 mM biotin before use

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

50, 160, 500, 1600 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9: sodium azide (SA, 1 µg/plate) for TA 100 and TA 1535; 2-nitrofluorene (2NF, 1 µg/plate) for TA 98 and TA 1537; cumene hydroperoxide (CHP, 100 µg/plate for plate incorporation and 25 µg/plate for preincubation) for TA 102;

Controls 2

Positive control substance:

other: + S9: 2-aminoanthracene (2-AA, 4 µg/plate for TA 102 or 2 µg/plate for remaining strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
1. in agar (plate incorporation)

DURATION
- Exposure duration: 72 h

2. preincubation

DURATION
- Preincubation period: 1 h
- Exposure duration: 72 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments including 1 plate incorporation and 1 preincubation test


DETERMINATION OF CYTOTOXICITY
- Method: other: thinning of the background lawn of non-revertant bacteria, appearance of micro-colonies and/or reduction in the number of revertant colonies on the test plates comparised to the solvent control plates

Evaluation criteria:

The test was considered to be valid when the following criteria were met:
1. negative and positive control data were consistent and within the range of historical control data (see table 4)
2. positive controls revealed a marked increase over the concurrent solvent controls
3. the evaluation was not restricted by loss of plates (e.g. through contamination)

The test material may be considered mutagenic in this test system if all of the following criteria were met:
1. dose-related increases in the number of revertants at one or more test points
2. reproducible increases in revertants between replicate plates
3. statistically significance in the increases of revertants
4. increases count more than twice the corresponding solvent control values

The test material may be considered non-mutagenic in this test system when no increases in the number of revertants is observed which exceed 1.5 times the solvent control values at any test point. Sporadically ocurring statistically significanct increases in the number of revertants which were not dose-related will usually be considered incidental and not relevant for the evaluation.

Increases between 1.5 and 2 fold compared to the respective solvent controls meeting the other criteria for a positive result were considered to demonstrate weak mutagenicity.

Statistics:

Statistical analyses were performed with the SAS (R) procedures version 8.1 (SAS Institute Inc., Cary, North Carolina 27513, USA).
In detail, the number of revertant colonies at each treatment test point were compared to the corresponding solvent control values using the Analysis of Variance test. Statistically significant differences were further evaluated via Dunnett´s test to determine the statistical significance of increases and decreases in the number of revertant colonies for each set of triplicates.

Results and discussion

Test results

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: no but tested up to limit concentrations (a small reduction in the number of revertants was induced in TA 100 (5000 µg: -15% (preincubation assay, +S9) and TA 1535 (160 µg: -28%; 1600 µg: -46%; 5000 µg: -30% (plate incorporation assay, + S9))

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test item did not induce toxicity within the conducted preliminary toxicity test as evaluated by large reductions in the number of revertants or poor growth of the background lawn of non-revertant bacteria either in the absence or presence of S9 mix (see table 1).

COMPARISON WITH HISTORICAL CONTROL DATA:
The solvent and positive control values were acceptable and compatible with the historical control values (slight increases have been determined in number of revertants for TA 102 in the plate incorporation (-S9: 470 ± 6 vs 409) and preincubation test (+S9: 460 ± 4 vs 432) and for TA 1535 in the plate incorporation assay after treatment with sodium azide (-S9: 1063 ± 83 vs 908), see table 4).

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Test results of the preliminary toxicity test (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard Deviation)
		Frameshift type
		TA98
–	0	36.0 ± 1.7
–	50	30.3 ± 3.1
–	160	28.7 ± 5.1
–	500	33.0 ± 6.0
–	1600	34.3 ± 2.9
–	5000	34.3 ± 7.0
Positive controls, –S9	Name	2NF
	Concentrations (μg/plate)	1
	Mean No. of colonies/plate (average of 3 ± SD)	499.7 ± 88.3
+	0	48 ± 3
+	50	48.3 ± 5.5
+	160	43.3 ± 2.9
+	500	40.0 ± 6.6
+	1600	40.0 ± 1.0
+	5000	39.7 ± 6.1
Positive controls, +S9	Name	2AA
	Concentrations (μg/plate)	2
	Mean No. of colonies/plate (average of 3 ± SD)	682 ± 26.5
2NF = 2 Nitrofluorene
2AA = 2-Aminoanthracene
No statistical analysis was performed.

 

 

Table 2. Test results of main test 1 (plate incorporation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	210.3 ± 7.5	14.7 ± 1.5	469.7 ± 6.1	30.7 ± 7.6	14.0 ± 2.0
–	50	210.7 ± 7.8	15.7 ± 2.5	474.0 ± 3.0	33.3 ± 7.2	17.7 ± 2.5
–	160	206.0 ± 5.3	15.0 ± 1.0	480.0 ± 6.6	27.7 ± 0.6	18.0 ± 2.0
–	500	205.7 ± 3.8	16.3 ± 2.1	478.7 ± 5.7	27.0 ± 2.6	15.0 ± 2.0
–	1600	208.7 ± 9.3	15.0 ± 3.6	479.0 ± 6.2	26.0 ± 5.0	16.3 ± 2.3
–	5000	208.3 ± 2.9	12.3 ± 1.5	483.7 ± 5.5	32.7 ± 3.1	15.7 ± 2.5
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	100	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1136.7 ± 60.8	1063 ± 82.5	968.3 ± 78.4	749.7 ± 100.5	202.0 ± 7.9
+	0	213.7 ± 3.8	20.3 ± 2.9	405.3 ± 1.5	43.3 ± 2.1	16.7 ± 1.5
+	50	213.3 ± 3.1	19.0 ± 1.0	399.7 ± 6.8	41.0 ± 2.0	20.3 ± 0.6
+	160	215.7 ± 3.8	14.7* ± 1.5	401.3 ± 10.2	44.0 ± 2.0	19.7 ± 0.6
+	500	202.0 ± 5.3	17.0 ± 2.6	405.0 ± 11.5	40.3 ± 2.5	18.7 ± 0.6
+	1600	208.0 ± 11.1	11.0 ± 1.7	406.7 ± 9.7	42.0 ± 3.6	18.7 ± 2.1
+	5000	216.7 ± 4.2	14.3* ± 3.1	405.7 ± 4.7	43.0 ± 1.7	19.0 ± 1.7
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	838.0 ± 71.4	276.0 ± 80.0	1030.3 ± 102.6	597.0 ± 25.6	366.7 ± 34.0
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

Table 3. Test results of main test 2 (preincubation)
With or without S9-Mix	Test substance concentration	Mean number of revertant colonies per plate
	(μg/plate)	(average of 3 plates ± Standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	TA102	TA98	TA1537
–	0	214.7 ± 4.0	18.0 ± 3.0	403.7 ± 3.1	36.0 ± 1.7	15.7 ± 1.2
–	50	213.3 ± 6.4	17.3± 0.6	405.0 ± 5.6	30.3 ± 3.1	18.3 ± 0.6
–	160	213.7 ± 6.0	18.3 ± 3.5	407.7 ± 2.3	28.7 ± 5.1	18.3 ± 1.5
–	500	213.0 ± 1.7	21.3 ± 4.2	403.7 ± 1.5	33.0 ± 6.0	19.0 ± 1.0
–	1600	211.0 ± 11.5	21.3 ± 3.2	406.7 ± 4.9	34.3 ± 2.9	18.3 ± 2.1
–	5000	212.3 ± 6.5	17.0 ± 1.7	411.3 ± 13.7	34.3 ± 7.0	17.0 ± 1.0
Positive controls, –S9	Name	SA	SA	CHP	2NF	2NF
	Concentrations (μg/plate)	1	1	25	1	1
	Mean No. of colonies/plate (average of 3 ± SD)	1103.0 ± 25.5	596.3 ± 25.5	1158.0 ± 33.2	499.7 ± 88.3	338.7± 22.7
+	0	203.7 ± 7.5	11.7 ± 1.2	459.7 ± 4.2	48.0 ± 3.0	18.7 ± 1.2
+	50	206.0 ± 12.2	17.0 ± 1.0	460.0 ± 2.6	48.3 ± 5.5	20.0 ± 1.0
+	160	213.7 ± 4.7	16.3 ± 4.2	456.7 ± 4.5	43.3 ± 2.9	19.0 ± 1.0
+	500	207.7 ± 6.8	15.7 ± 2.1	455.3 ± 3.8	40.0 ± 6.6	18.0 ± 1.7
+	1600	188.7 ± 3.1	15.0 ± 4.0	458.3 ± 2.5	40.0 ± 1.0	19.0 ± 1.0
+	5000	173.3** ± 9.5	17.7 ± 2.3	459.7 ± 4.2	39.7 ± 6.1	19.0 ± 1.0
Positive controls, +S9	Name	2AA	2AA	2AA	2AA	2AA
	Concentrations (μg/plate)	2	2	4	2	2
	Mean No. of colonies/plate (average of 3 ± SD)	574.7 ± 30.2	231.7 ± 18.9	1140.7 ± 98.8	682.0 ± 26.5	210.3 ± 16.0
SA = sodium azide
CH = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene
* = statistically significant at 5% level
* * = statistically significant at 1% level
Otherwise not statistically significant at 5% level (positive controls were not included)

 

 

Table 4. Historical control values
Strain	Treatment (µg/mL)	S9 mix		Number of revertant colonies/plate				Number of plates
				Mean	Standard Deviation	Minimum	Maximum
TA 102	solvent	-		305	40	249	409	114
	solvent	+		321	52	224	432	117
	CHP (25)	-		1262	346	836	2487	54
	CHP (100)	-		1252	258	903	2000	48
	2AA (4)	+		1076	197	500	1522	105
TA 100	solvent	-		144	33	91	221	162
	solvent	+		143	35	90	234	168
	SA (1)	-		1006	196	578	1460	150
	2AA (2)	+		1007	362	123	2163	156
TA 98	solvent	-		51	16	19	82	159
	solvent	+		61	17	21	97	162
	2NF (1)	-		658	247	216	1189	147
	2AA (2)	+		851	284	411	1622	150
TA 1537	solvent	-		15	5	5	28	111
	solvent	+		19	7	7	43	117
	2NF (1)	-		357	154	121	919	99
	2AA (2)	+		301	202	67	1121	105
TA 1535	solvent	-		20	8	7	38	117
	solvent	+		18	7	7	42	123
	SA (1)	-		541	169	206	908	105
	2AA (2)	+		272	129	110	648	111
SA = sodium azide
CHP = cumene hydroperoxide
2NF = 2-nitrofluorene
2AA = 2-Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative