3-(6,6-dimethylbicyclo[3.1.1]hept-2-en-2-yl)-2,2-dimethylpropanenitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The genetic toxicity of the test substance, Pinyl Nitrile was assessed and considered to be non-mutagenic according to OECD 471 using the Bacterial Reverse Mutation method.

The genotoxic potential of the test substance, Pinyl Nitrile was also assessed according to OECD Test Guideline 473 using an In Vitro Mammalian Chromosome Aberration method. The test item, Pinyl Nitrile, did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system, in either of two separate experiments. The test item was, therefore, considered to be non-clastogenic to Chinese Hamster Lung (CHL) or V79 Cells in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 April 2016 to 28 October 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Refer to main study report

GLP compliance:

yes

Remarks:

Refer to main study report

Type of assay:

other: In Vitro Mammalian Chromosomal Aberration Assay in Chinese Hamster Lung (CHL) or V79 Cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL- Source and lot/batch No.of test material: International Flavors & Fragrances Inc. Lot/Batch No.SM15077102- Expiration date of the lot/batch: 22 July 2016STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: 2 to 8° C, protected from light- Solubility of the test substance in the solvent/vehicle: Soluble in DMSO at a concentration of approximately 500 mg/mLTREATMENT OF TEST MATERIAL PRIOR TO TESTING- Treatment of test material prior to testing: Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under filtered light.

Target gene:

chromosomes

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED- Source of cells: Sigma Aldrich- Suitability of cells: Stability of karyotype and morphology has made them suitable for genetic toxicity assays with low background aberrations- Cell cycle length, doubling time or proliferation index: Population doubling time of approximately 15 hours- Modal number of chromosomes: 22MEDIA USED- Type and identity of media including CO2 concentration if applicable: complete medium [Dulbecco's Modified Eagle Medium with GlutaMAX™ –I, 1 g/L D-Glucose, 110 mg/L Sodium Pyruvate containing 10% heat inactivated fetal bovine serum, 100 units/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL Amphotericin B]. The cultures were incubated under standard conditions (37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air).- Properly maintained: yes- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was purchased commercially from MolTox (Boone, NC)..

Test concentrations with justification for top dose:

In the preliminary toxicity assay, the doses tested were 0.2, 0.6, 2, 6, 20, 60, 200, 600, and 2000 µg/mL. The top dose tested, 2000 µg/mL, was the limit dose for this assay, as per OECD 473 (adopted 2014). Dose levels for the chromosomal aberration assay were based upon post-treatment toxicity (cell growth inhibition relative to the vehicle control). Doses tested were:Non-activation 4 hour: 5.0, 10, 20, 30, 35, 40, 45, 50, 55, and 60 µg/mLS9-activation 4 hour: 20, 40, 60, 80, 100, 120, 140, 160, 180, and 200 µg/mLNon-activation 20 hour: 0.5, 1.0, 2.5, 5.0, 10, 20, 30, 35, 40, 45, and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: DMSO was used as the vehicle based on the information provided by the Sponsor, the solubility of the test substance, and compatibility with the target cells. In a solubility test conducted at BioReliance, the test substance was soluble in DMSO at a concentration of approximately 500 mg/mL, the maximum concentration tested for solubility.

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 mediumDURATION- Exposure duration: CHO cells were exposed to the test and control articles for 4 and 20 hours without S9 and for 4 hours with S9, and rinsed. Cells were harvested 20 hours (±30 minutes) after initiation of treatment, which corresponds to 1.5 normal cell cycles.STAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: 1 in the preliminary toxicity assay; 2 in the chromosome aberration assayNUMBER OF CELLS EVALUATED: a minimum of 300 metaphase spreads from each dose level (150 per duplicate culture), whenever possibleDETERMINATION OF CYTOTOXICITY- Method: Cell growth indexOTHER EXAMINATIONS:- Determination of polyploidy: yes- Determination of endoreplication: yes

Evaluation criteria:

Toxicity induced by treatment was based upon inhibition of cell growth and was reported for the cytotoxicity and chromosome aberration portions of the study. The number and types of aberrations (structural and numerical) found, the percentage of structurally damaged cells in the total population of cells examined (percent aberrant cells), the percentage of numerically damaged cells in the total population of cells examined, and the average number of structural aberrations per cell (mean aberrations per cell) were calculated and reported for each treatment group. Chromatid and isochromatid gaps are presented in the data but were not included in the total percentage of cells with one or more aberrations or in the average number of aberrations per cell.A test article was considered positive if it induced a statistically significant and dose dependent increase in the frequency of aberrant metaphases (p

Statistics:

Statistical analysis of the percentage of aberrant cells was performed using the Fisher's exact test. The Fisher's test was used to compare pairwise the percent aberrant cells of each treatment group with that of the vehicle control. The Cochran-Armitage test was used to measure dose-responsiveness.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Definitive assay: Cytotoxicity (55 ± 5% cell growth inhibition relative to vehicle control), was observed at doses >/= 35 µg/mL in the non activated 4 and 20-hour exposure groups, and at doses >/= 100 µg/mL in the S9 activated 4-hour exposure group.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: The pH of the highest dose of test substance in treatment medium was 7.5.- Effects of osmolality: The osmolality of the test substance doses in treatment medium is acceptable because it did not exceed the osmolality of the vehicle by more than 120%.- Definition of acceptable cells for analysis: Metaphase cells with 22 ± 2 centromeres were examined under oil immersion RANGE-FINDING/SCREENING STUDIES: A preliminary toxicity test was conducted in order to determine cytotoxicity and to select doses for the chromosomal aberration assay.HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)- Positive historical control data: Historical positive control chromosomal aberration data for CHL or V79 cells is not available due to limited number of studies conducted.- Negative (solvent/vehicle) historical control data: Historical vehicle control chromosomal aberration data for CHL or V79 cells is not available due to limited number of studies conducted.ADDITIONAL INFORMATION ON CYTOTOXICITY:- Measurement of cytotoxicity used: reduction in cell growth index relative to the vehicle control

Conclusions:

Under the conditions of the assay described in this report, ES421 Pinyl Nitrile was concluded to be negative for the induction of structural and numerical chromosome aberrations in the non-activated and S9-activated test systems in the in vitro mammalian chromosome aberration test using Chinese Hamster Lung (CHL) or V79 cells.

Executive summary:

The test substance,ES421 Pinyl Nitrile, was tested to evaluate the potential to induce structural chromosomal aberrations using Chinese hamster lung (CHL) or V79 cells in both the absence and presence of an of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO)was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.2 to 2000 µg/mL, which was the limit dose for this assay, as per OECD 473 (adopted 2014). Cytotoxicity (greater than 50% reduction in cell growth index relative to the vehicle control) was observed at doses ≥60 µg/mL in the non‑activated 4 and 20-hour exposure groups, and at doses ≥200 µg/mL in the S9‑activated 4-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses ≥600 µg/mL in the non‑activated and S9-acitvated 4-hour exposure groups, and at 2000 µg/mL in the non‑activated 20-hour exposure group. Based upon these results, the doses chosen for the chromosome aberration assay ranged from 5 to 60 µg/mL for the non‑activated 4-hour exposure group, from 20 to 200 µg/mL for the S9-activated 4-hour exposure group, and from 0.5 to 50 µg/mL for the non-activated 20-hour exposure group.

In the chromosome aberration assay, cytotoxicity (55 ± 5% reduction in cell growth index relative to the vehicle control), was observed at doses ≥35 µg/mL in the non‑activated 4 and 20-hour exposure groups, and at doses ≥100 µg/mL in the S9‑activated 4-hour exposure group. The doses selected for evaluation of chromosome aberrations were 10, 20, and 35 µg/mL for the non‑activated 4-hour exposure group; 20, 40, and 100 µg/mL for the S9‑activated 4-hour exposure group; and 5, 20, and 35 µg/mL for the non‑activated 20-hour exposure group.

No significant or dose‑dependent increases in structural or numerical (polyploid or endoreduplicated cells) aberrations were observed in treatment groups with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicateES421 Pinyl Nitrilewas negative for the induction of structural and numerical chromosome aberrations in the presence and absence of the exogenous metabolic activation systemin thein vitroMammalian Chromosomal Aberration Assay inChinese Hamster Lung (CHL) or V79 Cells.

Reference 2

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:

09 March 2016 to 27 October 2016

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

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella strains contain mutations in the histidine operon, thereby imposing a requirement for histidine in the growth medium. These strains contain the deep rough (rfa) mutation, which deletes the polysaccharide side chain from the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene, which codes 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). Tester strains TA98 and TA100 contain the R-factor plasmid, pKM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non-R-factor parent strains. pKM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system, which is normally present in these organisms. The tester strain Escherichia 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 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Initial toxicity-mutation assay: 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for all test conditionsConfirmatory mutagenicity assay: 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate for all test conditions

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (test article, 2- nitrofluorene, 9- aminoacridine, methylmethanesulfonate, 2- aminoanthracene); water (sodium azide)- Justification for choice of solvent/vehicle: DMSO was the vehicle of choice based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at a concentration of approximately 500 mg/mL in the solubility test conducted at BioReliance.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Exposure duration: 48 to 72 hoursNUMBER OF REPLICATIONS: 2 in the initial-toxicity mutation assay; 3 in the confirmatory mutagenicity assayNUMBER OF CELLS EVALUATED: >/= 0.3 x 10^8 cells/plateDETERMINATION OF CYTOTOXICITY- Method: other: Counting of revertant colony numbers and evaluation of the condition of the bacterial background lawn.

Evaluation criteria:

The revertant colony numbers were determined for each plate (counted either manually or by automatic colony counter). The mean and standard deviation of the number of revertants per plate were calculated and reported.For the test article to be evaluated positive (mutagenic), it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article as specified below:Strains TA1535 and TA1537Data sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value.Strains TA98, TA100 and WP2 uvrAData sets were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Statistics:

According to the test guidelines, the biological relevance of the results is the criterion for the interpretation of the results, and a statistical evaluation of the results is not regarded as necessary.

Key result

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

Remarks:

Confirmatory Mutagenicity Assay: Toxicity was observed beginning at 1500 µg per plate with tester strain TA100 in the absence of S9 activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):negativeAll criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, ES421 Pinyl Nitrile did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9.

Executive summary:

The test substance, ES421 Pinyl Nitrile, was tested to evaluate its mutagenic potential by measuring its ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system. Dimethyl sulfoxide (DMSO) was used as the vehicle.

In the initial toxicity-mutation assay, the dose levels tested were 1.50, 5.00, 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate. No definitive background lawn toxicity was observed. Precipitate was observed at 5000 µg per plate with all conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Based upon these results, the maximum dose tested in the confirmatory mutagenicity assay was 5000 µg per plate.

In the confirmatory mutagenicity assay, the dose levels tested were 15.0, 50.0, 150, 500, 1500 and 5000 µg per plate.  Precipitate was observed at 5000 µg per plate with all conditions. Toxicity was observed beginning at 1500 µg per plate with tester strain TA100 in the absence of S9 activation. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

All criteria for a valid study were met as described in the protocol. These results indicate ES421 Pinyl Nitrile was negative for the ability to induce reverse mutations at selected loci of several strains ofSalmonella typhimuriumand at the tryptophan locus ofEscherichia colistrain WP2 uvrA in the presence and absence of an exogenous metabolic activation system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.

The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

The in vitro gene mutation study (Ames) was designed to assess the mutagenic potential of the test material using a bacterial test system. The study was based on the in vitro technique described by Ames and his co-workers and Garner et al. In this study mutagenic activity is assessed by exposing histidine auxotrophs of Salmonella typhimurium and tryptophan auxotrophs of Escherichia coli to various concentrations of the test material. This method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test".

 

The Salmonella strains used in the test are incapable of synthesising histidine and are therefore dependent for growth on an external source of this particular amino acid. When exposed to a mutagenic agent, these bacteria may undergo a reverse mutation to histidine independent form which are detected by their ability to grow on a histidine deficient medium. Using various strains of this organism, revertants produced after exposure to a chemical mutagen may arise as a result of base-pair substitution in the genetic material (miscoding) or frame-shift mutation in which genetic material is added or deleted. In order to make the bacteria more sensitive to mutation by chemical and physical agents, several additional strains have been introduced. These include a deletion though the excision repair gene (uvrB- Salmonella strains) which renders the organism incapable of DNA excision repair and deep rough mutation (rfa) which increases the permeability of the cell wall. A mutant strain of E. coli (WP2uvrA-) which requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence was used to complement the salmonella strains. This strain also has a deletion in the excision repair gene (uvrA-). Since many compounds do not exert a mutagenic effect until they have been metabolised by enzyme systems not available in the bacterial cell, the test material and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre-treated with a compound known to induce an elevated level of these enzymes.

From the results of the Ames test, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested, with any dose of the test item, either with or without metabolic activation. The test item, IFF 213 (Floriane) was therefore considered to be non-mutagenic in vitro.

 

The purpose of the chromosome aberration study was to assess the potential chromosomal mutagenicity of the test item, on the metaphase chromosomes of normal human lymphocytes. The detection of structural chromosomal aberrations in cultured mammalian cells supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). Human peripheral blood lymphocytes are recognized in the OECD 473 guideline as being a suitable cell line for the Mammalian Chromosome Aberration Test. Numerical and structural chromosome aberrations are implicated in the pathology of neoplasia (Radmanet al., 1982; Cairns, 1981) and also occur in a high proportion of spontaneous abortions and abnormal live births (Chandley, 1981). Furthermore, most carcinogens are capable of inducing such changes in chromosome fidelity. Metaphase analysis in vitro involves recording such structural and numerical aberrations in the chromosomes of exposed cells. Many of these changes are lethal to the cells in which they occur and are therefore not of heritable significance. However, it is assumed that agents capable of inducing gross chromosomal changes also induce more subtle changes (translocations, inversions and small deletions) which are not cell lethal, and therefore represent a hazard. The ability to induce chromosome aberrations also correlates well with the induction of gene mutations (Hollsteinet al., 1979). This study was designed to be compatible with OECD Guidelines for Testing of Chemicals No. 473 "In Vitro Mammalian Chromosome Aberration Test".

 

It was established in the chromosome aberration test that the test item was toxic to human lymphocytes but did not induce any statistically significant increases in the frequency of cells with aberrations. The test item, IFF 213 (Floriane) was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Justification for selection of genetic toxicity endpoint
The study was conducted in vitro, in an appropriate test species and according to internationally recognised guidelines.

Justification for classification or non-classification

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

To this end, the test substance has been assessed according to internationally recognized guidelines in two in vitro tests. Firstly, an in vitro gene mutation study in bacteria (Ames test) and secondly an in vitro mammalian chromosome aberration study.

In the in vitro gene mutation study in bacteria (Ames) no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains tested with any dose of the test item, either with or without metabolic activation.

In the in vitro mammalian chromosome aberration study, the test item did not induce any statistically significant increases in the frequency of cells with chromosome aberrations, either with or without metabolic activation and was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Based on two negative results in vitro, the test item is considered non-mutagenic.