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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable in vitro test for genetic toxicity were available (Amen, Mouse Lympoma assay, Micronucleaus test). All three test were negative.

Link to relevant study records

Reference

Reference 1

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

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

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 / cell type:

S. typhimurium TA 97

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 102

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

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

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

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

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

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

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

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

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”.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

several (see 'Overall remarks')

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

TK locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

0.04 µL/mL – 0.0003 µL/mL (with metabolic activation) and 1.25 µL/mL to 0.01 µL/mL (without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: standard vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4h and 24h (experiment 2, without S9)

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Cell Numbers
Cell numbers in suspension were determined using an aliquot of the well homogenised suspension whose cell number was determined in a cell counter.

Colonies
Colonies were counted manually. In accordance with their size, the colonies were classified into two groups. The colony size distribution was determined in the controls and at all evaluated concentrations of the test item. Criteria to determine colony size were the absolute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).
All generated data of each experiment (test item, solvent control and positive control) were recorded in the raw data.

The test item is considered to have mutagenic effects if:
the induced mutation frequency reproducibly exceeds a threshold of 126 colonies1 per 106 cells above the corresponding solvent control.
the relative increase of the mutation frequency shows a dose relationship.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
Results of test groups are generally rejected if the relative total growth is less than 10% of the solvent control.
The biological relevance of the results was always considered first. Appropriate statistical methods were used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data was also taken into consideration.
Statistical significance was confirmed by means of the non-parametric χ2 test (chapter Statistical Analysis, page 62). However, both biological and statistical significance were considered together.

Statistics:

χ2 test

Species / strain:

mouse lymphoma L5178Y cells

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

Conclusions:

In a GLP-study according to OECD Test Guideline 476, the registered substance was negative in the L5178Y/TK+/- Mouse Lymphoma Assay.

Executive summary:

The test article, CARDOLITE NC-513, was tested in the L5178Y/TK^+/-Mouse Lymphoma Mutagenesis Assay in the absence and presence of metabolic activation with a 4hour exposure. The mutagenesis assay was used to evaluate the mutagenic potential of the test article.

First, a pre-experiment was performed in order to determine a potential cytotoxic effect.

The mutagenicity assay was performed in in two independent experiments (experiment I and II), using two parallel cultures each based on the results of the pre-experiment. The first main experiment (experiment I) was performed with and without metabolic activation (liver enzyme S9 fraction) and a treatment period of 4 h. The second experiment (experiment II) was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration (5 µL/mL) applied was chosen with regard to the solubility of the test item in DMSO. In the pre-experiment a strong cytotoxic effect was detected in the concentrations from 1.25 µL/mL to 0.08 µL/mL in the approach with metabolic activation. In the approach without metabolic activation a cytotoxic effect was detected at the concentration of 0.63 µL/mL. Based on the results of the pre-experiment, the concentrations in experiment I and II were re-adjusted to 0.04 µL/mL – 0.0003 µL/mL (with metabolic activation) and 1.25 µL/mL to 0.01 µL/mL (without metabolic activation).

The tested concentrations are described in table Test Item-a. The evaluated experimental points and the results are summarised in table 3-a and 3-b.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

In conclusion, it can be stated that in the mutagenicity test described and under the experimental conditions reported, the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Therefore, CARDOLITE NC-513 is considered to be “non-mutagenic under the conditions of the mouse lymphoma assay”.

.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

yes

Remarks:

some minor not critical deviations

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

preliminary test: 0.04 - 5 µl/ml in culture
Main experiments (without and with S9): 0.04, 0.02 and 0.01 µL/mL nominal

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Well established solvent for this test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
- Exposure duration:
with S9: 4 ± 1 hrs
without S9 (short): 4 ± 1 hrs
without S9 (extended): 1.5 - 2 cell cycles

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 2000 binucleated cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index

Cytokinesis was blocked with Cytochalasin B

Evaluation criteria:

The test item is considered to have no genotoxic effects if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory’s historical control data of the solvent control
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

The test item is considered to have genotoxic effects if:
- the number of micronucleated cells in all evaluated dose groups is above the range of the historical laboratory control data
- either a concentration-related increase of micronucleated cells or a statistically significant increase in the number of cells containing micronuclei is observed

Statistics:

Fisher’s exact test

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In a GLP-study according to OECD test guideline 487, the registered substance did not exhibit clastogenic or aneugenic activity in cultured human peripheral blood lymphocytes and is therefore considered negative.

Executive summary:

The genotoxic potential of CARDOLITE NC-513 with the In VitroMammalian Cell Micronucleus Test following OECD 487 was determined.

This study was performed in order to evaluate the mutagenic potential of CARDOLITE NC-513 to induce formation of micronuclei in human lymphocytes. The lymphocytes, in whole blood culture, were stimulated to divide by addition of phytohaemagglutinin and exposed to the test item both in absence and presence of an exogenous metabolic activation system (liver S9 mix from male rats, treated with Aroclor 1254). The proportion of cells containing micronuclei was determined.

Two independent experiments were performed. In each experimental group, all cell cultures were set up in duplicate. In order to assess the toxicity of the test item to cultured human lymphocytes, the cytokinesis-block proliferation index was calculated in a pre-experiment. The test item showed high cytotoxicity down to a concentration of 0.08 µL/mL in both experimental parts (with and without metabolic activation). Therefore, experiment I and experiment II were performed with the following concentrations of the test item: 0.04, 0.02, 0.01, 0.005, 0.003, 0.001, 0.0006 and 0.0003 µL/mL nominal. Again, the cytokinesis-block proliferation index was calculated and on the basis of this data, the following concentrations were selected for micronuclei scoring:

Experiment I

Without S9 mix / 4 ± 1 hours exposure: 0.04, 0.02 and 0.01 µL/mL nominal

With S9 mix / 4 ± 1 hours exposure: 0.04, 0.02 and 0.01 µL/mL nominal

Experiment II

Without S9 mix / 22 ± 2 hours exposure: 0.04, 0.02 and 0.01 µL/mL nominal

With S9 mix / 4 hours exposure: 0.04, 0.02 and 0.01 µL/mL nominal

All positive control compounds – at least in the higher concentrations - caused large, statistically significant increases in the proportion of micronucleated cells, demonstrating the sensitivity of the test system.

In conclusion, under the experimental conditions reported, CARDOLITE NC-513 does not induce the formation of micronuclei in human lymphocytesin vitro.

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance did not show genotoxic potential in an Ames test, in a Mammalian Cell Micronucleus Test and in a Mammalian Cell Gene Mutation Test.

Based on these in vitro results, the substance is considered to be not genotoxic and does not require classification according to Regulation (EC) No. 1272/2008.