Hexane-1,3,6-tricarbonitrile

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.01.10-2022.07.06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

mouse lymphoma thymidine kinase locus (tk+/-)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

An appropriate quantity of S9 fraction was thawed and mixed with a co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. In 100 mM sodium phosphate buffer pH 7.4, the following concentrations were achieved: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP. During the experiment the S9 mix was stored on ice.

Test concentrations with justification for top dose:

Criteria to determine the highest concentration are cytotoxicity, solubility in the test system, and changes of pH or osmolality. Cytotoxicity was determined with and without metabolic activation.
The test item was investigated at the following concentrations:
STE(-): 1.25, 2.5, 5.0, 7.5, 10 mM
STE(+): 1.25, 2.5, 5.0, 7.5, 10 mM
Solvent and negative controls were tested in duplicate.

Vehicle / solvent:

DSMO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

methylmethanesulfonate

other: ethylmethanesulfonate

Details on test system and experimental conditions:

accordant with the OECD TG 490

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Growth inhibition was observed in STE(-). The relative total growth (RTG) was 61% for highest concentration evaluated (10 mM) . No growth inhibition was observed in STE(+)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

In conclusion, in this in vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/tk+/-) in L5178Y Mouse Lymphoma Cells, under the experimental conditions reported, the test item Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic.

Executive summary:

The test item Hexane-1,3,6-tricarbonitrile was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus (tk^+/-) using the L5178Y cell line.

For the short-term experiments (STE), the test item was dissolved in DMSO and incubated with cells for 4 hours. The STEs were performed independently: with (STE⁽⁺⁾) and without (STE^(-)) metabolic activation.

The test item was investigated at the following concentrations:

STE^(-):    1.25, 2.5, 5.0, 7.5, 10 mM

STE⁽⁺⁾     1.25, 2.5, 5.0, 7.5, 10 mM

No precipitation of the dissolved test item was noted in any experiment.

Growth inhibition was observed in STE^(-). The relative total growth (RTG) was 61% for highest concentration evaluated (10 mM) (Table 3).

No growth inhibition was observed in STE⁽⁺⁾ (Table 6).

No biologically relevant increase of mutants was found in any experiments. The global evaluation factor (GEF) was not exceeded by the induced mutant frequency.

Ethyl methanesulfonate (EMS), Methyl methanesulfonate (MMS), and Benzo[a]pyrene (B[a]P) were used as positive controls and demonstrated distinct and biologically relevant responses in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potentially clastogenic effects.

In conclusion, in this in vitro Mammalian Cell Gene Mutation Assay (Thymidine Kinase Locus/tk^+/-) in L5178Y Mouse Lymphoma Cells, under the experimental conditions reported, the test item Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.01.10-2022.09.08

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)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) [11], [12] for three consecutive days by oral route. The preparation was performed according to Ames et al. [13].
The following quality control determinations are performed:
a) Biological activity in the Salmonella typhimurium assay using 2-aminoanthracene and benzo[a]pyrene
b) Sterility Test
A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at  -75°C.
The protein concentration in the S9 preparation (pre-experiment: Lot No. 191121, main experiment I: Lot No. 240622) was 35 and 32.2 mg/mL, respectively.

An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4. During the experiment the S9 mix was stored on ice.
The final concentration of S9 mix in the cultures is 5%.

Test concentrations with justification for top dose:

Duplicate cultures were treated at each concentration. The following concentrations were used in the main experiments:
Experiment I:
without and with metabolic activation: 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
Experiment II:
without metabolic activation: 0.25, 0.50, 1.0, 2.5, 5.0, 7.5 and 10 mM

The following concentrations were selected for the microscopic analyses of micronuclei frequencies. For main experiment I the maximum concentration of 10 mM was selected as highest concentration since no cytotoxicity or precipitation was observed. For main experiment II the highest concentration selected was based on cytotoxicity.
Experiment I with short-term exposure (4 h):
without and with metabolic activation: 5.0, 7.5 and 10 mM
Experiment II with long-term exposure (24 h):
without metabolic activation: 1.0, 2.5, 5.0 and 7.5 mM

Vehicle / solvent:

1% DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

colchicine

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:

Complete Culture Medium
MEM medium supplemented with:
10 % fetal bovine serum (FBS)
100 U/100 µg/mL penicillin/streptomycin solution
2 mM L-glutamine
2.5 µg/mL amphotericin
25 mM HEPES

Treatment Medium (short-term exposure)
Complete culture medium without FBS.

After Treatment Medium / Treatment Medium (long-term exposure)
Complete culture medium with 10% FBS and 1.5 µg/mL cytochalasin B.

A pre-experiment was conducted under identical conditions as described for the main experiment I. The following concentrations were tested only with S9 mix:
0.039, 0.078, 0,156, 0.313, 0.625, 1.25, 2.5, 5.0, 7.5 and 10 mM
The concentration of 10 mM was considered to be the highest test concentration used in this test system following the recommendation of the corresponding OECD testing guideline 487

Duplicate cultures were treated at each concentration. The following concentrations were used in the main experiments:
Experiment I:
without and with metabolic activation: 0.5, 1.0, 2.5, 5.0, 7.5 and 10 mM
Experiment II:
without metabolic activation: 0.25, 0.50, 1.0, 2.5, 5.0, 7.5 and 10 mM
The following concentrations were selected for the microscopic analyses of micronuclei frequencies. For main experiment I the maximum concentration of 10 mM was selected as highest concentration since no cytotoxicity or precipitation was observed. For main experiment II the highest concentration selected was based on cytotoxicity.
Experiment I with short-term exposure (4 h):
without and with metabolic activation: 5.0, 7.5 and 10 mM
Experiment II with long-term exposure (24 h):
without metabolic activation: 1.0, 2.5, 5.0 and 7.5 mM

Experiment I
Exponentially growing V79 cells were seeded into 25 cm2 cell culture flasks (two flasks per test group). Approx. 50 000 cells were seeded per cell culture flask, containing 5 mL complete culture medium (minimum essential medium supplemented with 10% FBS). After an attachment period of approx. 48 h, the complete culture medium was removed and subsequently the test item was added to the treatment medium in appropriate concentrations. The cells were incubated with the test item for 4 h in presence or absence of metabolic activation. At the end of the incubation, the treatment medium was removed and the cells were washed twice with PBS. Subsequently, the cells were incubated in complete culture medium + 1.5 µg/mL cytochalasin B for 20 h at 37°C

Experiment II
If negative or equivocal results are obtained, they should be confirmed using continuous treatment (long-term treatment) without metabolic activation. Approx. 50 000 exponentially growing V79 cells were seeded in 25 cm2 cell culture flasks in absence of metabolic activation. After an attachment period of approx. 48 h the test item was added in complete culture medium. 1 h later 1.5 µg/mL cytochalasin B were added and the cells were incubated for 23 h at 37°C. At the end of the treatment the cell culture medium was removed and the cells were prepared for microscopic analysis.

Evaluation criteria:

All slides, including those of positive and negative controls were independently coded before microscopic analysis. For each experimental point, at least 2000 binucleated cells per concentration (1000 binucleated cells per slide) were analysed for micronuclei according to the criteria of Fenech [7], i.e. clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges. Mononucleated and multinucleated cells and cells with more than six micronuclei were not considered

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

it can be stated that during the study described and under the experimental conditions reported, the test item Hexane-1,3,6-tricarbonitrile did not induce structural and/or numerical chromosomal damage in Chinese hamster V79 cells.
Therefore, Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in this in vitro Mammalian Cell Micronucleus Test.

Executive summary:

In experiment I with and without metabolic activation no increase of the cytostasis above 30% was noted. In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to 2.5 mM. At 5.0 mM a cytostasis of 46% and at 7.5 mM a cytostasis of 66% was observed.

In the main experiments I and II with and without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

No statistically significant increase (p<0.05) of cells with micronuclei was noted in the dose groups of the test item evaluated in experiment I and II with and without metabolic activation.

The c² Test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions. No statistically significant increase in the frequency of micronucleated cells under the experimental conditions of the study was observed in experiment I and II (Table 14).

Negative, solvent and positive controls were included in each experiment. The micronucleated cell frequency of the negative and solvent controls were within the historical control limits of the negative/solvent controls. MMS (25 µg/mL) and CPA (5.0 µg/mL) were used as clastogenic controls and colchicine as aneugenic controls (0.08 and 2.0 µg/mL). They induced distinct and statistically significant increases of the micronucleus frequency. This demonstrated the validity of the assay.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022.01.10-2022.05.02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The S. typhimurium histidine (his) reversion system and the E. coli tryptophan (trp) reversion system

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:Eurofins Munich
- method of preparation of S9 mix :The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)The protein concentration in the S9 preparation was 35.0 mg/mL

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment

5.0 μL/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate

Evaluation criteria:

Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs
in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high
- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control [11].
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

not applicable

True 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:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True 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:

not applicable

True negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

It can be stated that during the described mutagenicity test and under the experimental conditions reported, Hexane-1,3,6-tricarbonitrile did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Hexane-1,3,6-tricarbonitrile is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Hexane-1,3,6-tricarbonitrile was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II, 60 min at 37 °C) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
No toxic effects of the test item were noted in any of the five tester strains used up to the highest dose group evaluated with and without metabolic activation in experiment I and II.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Hexane-1,3,6-tricarbonitrile at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available