Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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:

Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Cas Number:

68413-24-1

Molecular formula:

C24H36O2

IUPAC Name:

Cashew (Anacardium occidentale) Nutshell Extract, Decarboxylated, Distilled, oligomerisation products with 1-chloro-2,3-epoxypropane

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): CARDOLITE NC-513
- Physical state: liquid
- Batch no.: GH-0129
- Storage condition of test material: room temperature 20 ± 5 °C, keep away from humidity
- Other: the test item was previously considered as 'Cashew, nutshell liq., oligomeric reaction products with 1-chloro-2,3-epoxypropane' (EC number 500-210-7).

Method

Target gene:

All Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chi) and biotin (bio) genes (bacteria require biotin for growth).The tester strains TA 98 and TA 100 contain the R-factor plasmid, pl(M101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. plcM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms (6), (9).The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagens which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

50 - 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO is an established solvent

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation test

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other:
revertant colony numbers and the inhibition of the background lawn

Evaluation criteria:

The colonies were counted visually, the numbers were recorded. A spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants less mean spontaneous revertants) were also calculated.
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor  2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

none applied

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 97a

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Mean Revertants First Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	106	107	20	18	80	78	134	138	18	17
	sd	9	8	2	2	11	10	7	4	2	1
DMSO	Mean	110	115	18	17	75	96	133	135	16	16
	sd	6	4	2	2	14	10	8	8	2	2
Positive Controls*	Mean	505	542	196	211	553	570	551	569	210	214
	sd	52	23	11	12	11	37	28	30	7	9
	f(I)	4.59	4.71	10.89	12.41	6.91	5.94	4.14	4.21	11.67	13.38
5004 µg/pl.	Mean	120	126	16	17	84	92	126	134	14	18
	sd	7	11	1	2	9	6	8	5	3	3
	f(I)	1.09	1.10	0.89	1.00	1.12	0.96	0.95	0.99	0.88	1.13
1501 µg/pl.	Mean	116	125	14	17	78	92	123	136	17	18
	sd	2	2	3	1	6	5	4	4	2	2
	f(I)	1.05	1.09	0.78	1.00	1.04	0.96	0.92	1.01	1.06	1.13
500 µg/pl.	Mean	108	119	19	15	18	93	114	126	16	16
	sd	6	4	2	4	2	3	3	10	3	2
	f(I)	0.98	1.03	1.06	0.88	0.24	0.97	0.86	0.93	1.00	1.00
150 µg/pl.	Mean	113	117	18	18	18	86	135	138	17	18
	sd	4	2	2	1	2	3	5	4	2	3
	f(I)	1.03	1.02	1.00	1.06	0.24	0.90	1.02	1.02	1.06	1.13
50 µg/pl.	Mean	111	116	14	20	17	85	135	124	15	17
	sd	4	2	1	1	2	12	7	8	1	1
	f(I)	1.01	1.01	0.78	1.18	0.23	0.89	1.02	0.92	0.94	1.06

Mean Revertants Second Experiment

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	105	124	20	17	86	90	133	123	17	14
	sd	6	3	6	2	3	9	10	8	2	2
DMSO	Mean	107	117	20	18	90	93	133	125	16	16
	sd	11	6	3	2	14	3	5	6	3	1
Positive Controls*	Mean	524	477	205	220	481	487	516	450	188	211
	sd	38	35	9	13	22	40	34	8	9	16
	f(I)	4.90	4.08	10.25	12.22	5.59	5.24	3.88	3.60	11.06	13.19
5041 µg/pl.	Mean	116	115	14	13	96	102	134	130	17	14
	sd	4	2	5	4	5	8	5	10	2	3
	f(I)	1.08	0.98	0.70	0.72	1.07	1.10	1.01	1.04	1.06	0.88
2521 µg/pl.	Mean	116	115	15	19	101	91	142	140	14	14
	sd	3	5	5	8	8	14	6	5	1	2
	f(I)	1.08	0.98	0.75	1.06	1.12	0.98	1.07	1.12	0.88	0.88
1261 µg/pl.	Mean	113	106	14	17	88	89	140	136	14	16
	sd	6	6	6	4	8	8	2	3	2	2
	f(I)	1.06	0.91	0.70	0.94	0.98	0.96	1.05	1.09	0.88	1.00
631 µg/pl.	Mean	114	111	11	18	82	89	125	134	16	13
	sd	3	5	3	2	9	2	5	7	2	1
	f(I)	1.07	0.95	0.55	1.00	0.91	0.96	0.94	1.07	1.00	0.81
316 µg/pl.	Mean	111	107	13	12	82	86	134	124	17	15
	sd	7	6	4	3	9	6	8	6	2	2
	f(I)	1.04	0.91	0.65	0.67	0.91	0.92	1.01	0.99	1.06	0.94

f(I) = increase factor

Applicant's summary and conclusion

Conclusions:

In a GLP study according to OECD Test Guideline 471 (Ames test), the registered substance was not mutagenic.

Executive summary:

The mutagenic potential of CARDOLITE NC-513 was determined with the Bacterial Reverse Mutation Test following OECD 471 and EU B.13/14. Two valid experiments were performed.

First Experiment:

Five concentrations of the test item, dissolved in dimethyl sulfoxide (DMSO) (ranging from 5004 to 50 µg/plate) were used. Five genetically manipulated strains ofSalmonella typhimurium(TA 97a, TA 98, TA 100, TA 102 and TA 1535) were exposed to the test item both in the presence and in the absence of a metabolic activation system (S9) for 48 hours, using the plate incorporation method.

None of the concentrations caused a significant increase in the number of revertant colonies in the tested strains. The test item didn’t show any mutagenic effects in the first experiment.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Second Experiment:

To verify the results of the first experiment, a second experiment was performed, using five concentrations of the test item (ranging from 5041 to 316 µg/plate) and a modification in study performance (pre-incubation method).

The test item didn’t show mutagenic effects in the second experiment, either.

No signs of toxicity towards the bacteria could be observed.

The sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range. All positive controls showed mutagenic effects with and without metabolic activation.

Under the conditions of the test, the test item did not show mutagenic effects towards Salmonella typhimurium ,strains TA 97a, TA 98, TA 100, TA 102 and TA 1535.

The test item CARDOLITE NC-513 is considered as “not mutagenic under the conditions of the test”.