Hexanamide, N-(2-hydroxyethyl)-3,5,5-trimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the in vitro Ames, chromosomal aberration and MLA assays, the test substance is not considered to be genotoxic.

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 (10%)

Test concentrations with justification for top dose:

17, 50, 167, 500, 1667 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility and acceptable solvent for use with Ames test.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar plate. Both direct plate incorporation method and preincubation method used

DURATION
- Preincubation period: 20 mins
- Exposure duration: Total exposure 3 days

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Condition of background lawn.

Evaluation criteria:

S. typhimurium strains TA 1535, TA 1537, and TA 98 and for E. coli WP2uvrA, 2 fold increase over mean concurrent vehicle control value.
S. typhimurium strain TA 100, a 1.5 fold increase over mean concurrent control value.

Statistics:

No

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Not required

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the first test, conducted by the Direct Plate Incorporation Method, toxicity to the bacteria was observed as a thinning of the background lawn of microcolonies, +/- a reduction in revertant colony numbers. This observation was made at the highest concentration of 5000 µg per plate in all strains in the absence of S9 mix and in strains TA 1535, TA 1537 and TA 100 in the presence of S9 mix.

In the second test, conducted by the Pre-incubation Method, toxicity was observed in all the bacterial strains at the 2 highest concentrations of 1667 and 5000 µg per plate, in both the absence and the presence of S9 mix.

Remarks on result:

other: no mutagenic potential

Remarks:

all strains/cell types tested

Tables in attachment (background material).

Conclusions:

Under the conditions of the study, the substance was determined to be non-mutagenic in the reverse mutation assay with and without metabolic activation

Executive summary:

An in vitro study was conducted to investigate the potential of test substance to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2uvrA. Two independent tests were conducted on agar plates in triplicate in the absence and presence of an Aroclor 1254 induced rat liver S9 preparation and the co-factors required for mixed-function oxidase activity (S9 mix). The first test was conducted by the Direct Plate Incorporation Method, while the second test was conducted by the Pre-incubation Method. The test substance was dissolved and diluted in dimethylsulphoxide and was dosed at concentrations ranging from 17 to 5000 µg per plate in both the absence and the presence of S9 mix. The highest concentration represented the maximum concentration recommended by the guideline. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No evidence of mutagenic activity was obtained with any strain in either test. The highest concentrations of the test substance were toxic to the bacteria, especially in the second test based on Pre-incubation Method. No precipitation of the test substance was observed at any of the tested concentrations. Under the conditions of the study, the substance was determined to be non-mutagenic in the reverse mutation assay with and without metabolic activation (Riach, 2012).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

AUG12/OCT12

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Hams' F-10
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 (10%)

Test concentrations with justification for top dose:

Test 1, with and without metabolic activation: 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/mL
Test 2, with metabolic activation: 75, 100, 150, 200, 250, 300, 350 and 400 µg/mL
Test 2, without metabolic activation: 37.5, 50, 75, 150, 200, 300, 400, 500 and 600 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility and acceptable solvent for use with Chromosomal aberration Test

Untreated negative controls:

no

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: Test 1, 6 h with and without metabolic activation, Test 2, 6 h with metabolic activation, 22 h without metabolic activation
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Test 1 and 2) and 48 h (Test 2)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Geimsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 per concentration

DETERMINATION OF CYTOTOXICITY
- Method: Cell counts relative to vehicle controls

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes
- Other:

OTHER:

Evaluation criteria:

Comparison with historical data

Statistics:

No

Species / strain:

Chinese hamster Ovary (CHO)

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None
- Effects of osmolality: None
- Precipitation: with metabolic activation 156-5000 µg/mL, without metabolic activation 300-5000 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Test 1, with and without metabolic activation: 625-5000 µg/mL
Test 2, with metabolic activation: 300-400 µg/mL
Test 2, without metabolic activation: 150-600 µg/mL

Remarks on result:

other: no clastogenic potential

Remarks:

all strains/cell types tested

Tables in attachment (background material)

Conclusions:

Under the conditions of the study, the test substance did not induce structural chromosome aberrations in the Chinese hamster ovary cell line with and without metabolic activation

Executive summary:

AAn in vitro study was conducted to investigate the potential of test substance to induce chromosome aberrations in Chinese hamster ovary (CHO), according to OECD Guideline 473, in compliance with GLP. Two independent experiments were performed with Chinese hamster ovary cell cultures in duplicate in the presence and absence of S9 mix (exogenous source of metabolic activation). Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. The two tests were conducted with the following test concentrations: Test 1 (with and without metabolic activation): 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/mL; Test 2 (with metabolic activation): 75, 100, 150, 200, 250, 300, 350 and 400 µg/mL; Test 2 (without metabolic activation): 37.5, 50, 75, 150, 200, 300, 400, 500 and 600 µg/mL.

The S9 mix was a post-mitochondrial supernatant fraction prepared from the livers of adult, male rats treated with Aroclor 1254. The test concentrations Cultures, established 20-24 h before testing, were treated for 6 h in the presence and 6 h or 22 h in the absence of S9 mix. Cultures were then harvested at 24 h (Test 1 and 2) or 48 h (Test 2) post treatment. The test substance was found to be toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested up to the maximum permitted concentration of 5000 µg/mL in both the presence and absence of S9 mix (Test 1), where toxicity was noted at 625-5000 µg/mL in both the presence and absence of S9 mix. In Test 2, the test substance was tested up to 400 µg/mL in the presence of S9 mix and up to 600 µg/mL in the absence of S9 mix. Toxicity was noted in cultures treated with 300‑400 µg/mL (presence of S9 mix) and in cultures treated with 150‑600 µg/mL (absence of S9 mix). There was no evidence that the test substance induced structural chromosomal aberrations in either the presence or absence of S9 mix. The test substance also did not induce polyploidy in the absence of S9 mix in cultures harvested 48 h post treatment. Under the conditions of the study, the test substance did not induce structural chromosome aberrations in the Chinese hamster ovary cell line with and without metabolic activation (Murie, 2012).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec2012/Feb2013

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)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9 (10%)

Test concentrations with justification for top dose:

Assay 1, without metabolic activation: 10, 20, 40, 80, 120, 160, 200, 240 µg/mL
Assay 2, with metabolic activation: 20, 40, 80, 120, 160, 200, 240, 280 µg/mL
Assay 3, without metabolic activation: 25, 50, 75, 100, 125, 150, 175, 200 µg/mL
Assay 4, with metabolic activation: 160, 190, 220, 250, 280, 310, 340, 370 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and acceptable solvent for use with test

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

ethylmethanesulphonate

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Assays 1, 2 and 4: 4 h, Assay 3: 24 h

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RTG) - RTG represents the product of the growth in cell numbers post-reatment and the cloning efficiency at final
assessment, expressed relative to the mean of the vehicle control cultures.

Evaluation criteria:

IWGT published guidance.

Statistics:

Not required.

Key result

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: None
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Yes

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Assay 1: Definitive level of toxicity at 160 µg/mL (RTG = 13%)
Assay 2: 29% RTG at 280 µg/mL (above definitive range of 10-20%)
Assay 3: Definitive level of toxicity at 150 µg/mL (RTG = 11%) and 175 µg/mL (RTG = 16%)
Assay 4: 24 % RTG at 280 µg/mL and 9% RTG at 310 µg/mL. These levels met the stated criteria for a valid test.

Remarks on result:

other: non-mutagenic

Remarks:

all strains/cell types tested

Tables in attachment.

Conclusions:

Under the conditions of the study, the test substance did not induce gene mutations in the TK locus in mouse lymphoma L5178Y cells either in the presence or absence of metabolic activation.

Executive summary:

An in vitro study was conducted to investigate the potential of test substance to induce gene mutations at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cell line, according to OECD Guideline 476, in compliance with GLP. The test substance was formulated in dimethylsulphoxide. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). Preliminary cytotoxicity tests showed that the test substance was of a moderate order of toxicity, reducing cell growth over the range 33.3 to 333.3 µg/mL, above which concentrations were lethal.

Four independent mutation assays were conducted with the following test concentrations:

 

Assay No.	Presence or absence of S9	Treatment time (h)	Concentrations^†(µg/mL)
1	Absence	4	20, 40, 80, 120, 160
2	Presence	4	160, 200, 240, 280
3	Absence	24	75, 100, 125, 150, 175,
4	Presence	4	190, 220, 250, 280, 310

^†Concentrations plated-out for final assessment.

Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate. Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, all experiments were tested for dose-related trends in mutant fraction. No relevant evidence of mutagenic activity was obtained in any experiment. In the absence of S9 mix results were obtained at concentrations resulting in a critical level of toxicity (in the range 10% to 20% relative total growth) in both assays (4 h exposure period (Experiment 1) and 24 h exposure period (Experiment 3)). In the presence of S9 mix results were obtained in Experiment 4 at levels of toxicity (24% and 9% relative total growth) that met the stated criteria for a valid test. Under the conditions of the study, the test substance did not induce gene mutations in the TK locus in mouse lymphoma L5178Y cells either in the presence or absence of metabolic activation (Riach, 2013).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

An in vitro study was conducted to investigate the potential of test substance to induce gene mutations Salmonella typhimurium strains, according to OECD Guideline 471, in compliance with GLP. The test substance was tested for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and in Escherichia coli WP2uvrA. Two independent tests were conducted on agar plates in triplicate in the absence and presence of an Aroclor 1254 induced rat liver S9 preparation and the co-factors required for mixed-function oxidase activity (S9 mix). The first test was conducted by the Direct Plate Incorporation Method, while the second test was conducted by the Pre-incubation Method. The test substance was dissolved and diluted in dimethylsulphoxide and was dosed at concentrations ranging from 17 to 5000 µg per plate in both the absence and the presence of S9 mix. The highest concentration represented the maximum concentration recommended by the guideline. Concurrent positive controls demonstrated the sensitivity of the assay and the metabolising activity of the S9 mix. No evidence of mutagenic activity was obtained with any strain in either test. The highest concentrations of the test substance were toxic to the bacteria, especially in the second test based on Pre-incubation Method. No precipitation of the test substance was observed at any of the tested concentrations. Under the conditions of the study, the substance was determined to be non-mutagenic in the reverse mutation assay with and without metabolic activation (Riach, 2012).

Chromosomal aberration assay

An in vitro study was conducted to investigate the potential of test substance to induce chromosome aberrations in Chinese hamster ovary (CHO), according to OECD Guideline 473, in compliance with GLP. Two independent experiments were performed with Chinese hamster ovary cell cultures in duplicate in the presence and absence of S9 mix (exogenous source of metabolic activation). Dimethylsulphoxide was the vehicle and cyclophosphamide and methyl methanesulphonate were the positive controls used in both tests. The two tests were conducted with the following test concentrations:

- Experiment 1 (with and without metabolic activation: 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 µg/mL;

- Experiment 2 (with metabolic activation): 75, 100, 150, 200, 250, 300, 350 and 400 µg/mL;

- Experiment 2 (without metabolic activation): 37.5, 50, 75, 150, 200, 300, 400, 500 and 600 µg/mL.

Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). The test concentrations Cultures, established 20-24 h before testing, were treated for 6 h in the presence and 6 h or 22 h in the absence of S9 mix. Cultures were then harvested at 24 h (Experiment 1 and 2) or 48 h (Experiment 2) post treatment. The test substance was found to be toxic to Chinese hamster ovary cells in vitro in both the presence and absence of S9 mix. It was tested up to the maximum permitted concentration of 5000 µg/mL in both the presence and absence of S9 mix (Experiment 1), where toxicity was noted at 625-5000 µg/mL in both the presence and absence of S9 mix. In Experiment 2, the test substance was tested up to 400 µg/mL in the presence of S9 mix and up to 600 µg/mL in the absence of S9 mix. Toxicity was noted in cultures treated with 300‑400 µg/mL (presence of S9 mix) and in cultures treated with 150‑600 µg/mL (absence of S9 mix). There was no evidence that the test substance induced structural chromosomal aberrations in either the presence or absence of S9 mix. The test substance also did not induce polyploidy in the absence of S9 mix in cultures harvested 48 h post treatment. Under the conditions of the study, the test substance did not induce structural chromosome aberrations in the Chinese hamster ovary cell line with and without metabolic activation (Murie, 2012).

Mouse lymphoma assay (MLA):

An in vitro study was conducted to investigate the potential of test substance to induce gene mutations at the thymidine kinase (TK) locus in mouse lymphoma L5178Y cell line, according to OECD Guideline 476, in compliance with GLP. The test substance was formulated in dimethylsulphoxide. Tests were conducted both in the absence and in the presence of a post-mitochondrial supernatant fraction obtained from Aroclor 1254-induced livers of adult male rats and the co-factors required for mixed-function oxidase activity (S9 mix). Preliminary cytotoxicity tests showed that the test substance was of a moderate order of toxicity, reducing cell growth over the range 33.3 to 333.3 µg/mL, above which concentrations were lethal.

Four independent mutation assays were conducted with the following test concentrations:

- Experiment 1 (4 h, without metabolic activation): 20, 40, 80, 120, 160 µg/mL;

- Experiment 2 (4 h, with metabolic activation): 160, 200, 240, 280 µg/mL;

- Experiment 3 (24 h, without metabolic activation): 75, 100, 125, 150, 175 µg/mL and

- Experiment 4 (4 h, with metabolic activation): 190, 220, 250, 280, 310 µg/mL.

Positive control cultures were included, and the resultant mutant fractions from these provided the expected increase and proof of adequate recovery of ‘small’ type colonies. Duplicate cultures were carried through the experiments for each treatment point. Vehicle control cultures were also included and were tested in quadruplicate. Biological relevance was given to any increase in mutant fraction greater than 126 mutants per million above the concurrent control value. In addition, all experiments were tested for dose-related trends in mutant fraction. No relevant evidence of mutagenic activity was obtained in any experiment. In the absence of S9 mix results were obtained at concentrations resulting in a critical level of toxicity (in the range 10% to 20% relative total growth) in both assays (4 h exposure period (Experiment 1) and 24 h exposure period (Experiment 3)). In the presence of S9 mix results were obtained in Experiment 4 at levels of toxicity (24% and 9% relative total growth) that met the stated criteria for a valid test. Under the conditions of the study, the test substance did not induce gene mutations in the TK locus in mouse lymphoma L5178Y cells either in the presence or absence of metabolic activation (Riach, 2013).

Justification for classification or non-classification

Based on the results of the in vitro Ames, chromosomal aberration and MLA assays, the test substance does not warrant a classification for genotoxicity according to EU CLP (EC 1272/2008) criteria.