Isodecyl 3,5,5-trimethylhexanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key studies were conducted to internationally recognised testing guidelines and with GLP certification.

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

Study period:

13 April 2017 - 1 September 2017

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:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Isodecyl 3,5,5-trimethylhexanoate (CAS number 59231-35-5), batch number P7560, was a colourless liquid. It was received on 10 April 2017 and stored at 15-25°C, protected from light. Purity was stated as UVCB -100% (broad mixture of isomers) and the retest/expiry date was given as December 2018. The test article information provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor. Preliminary solubility data indicated that Isodecyl 3,5,5-trimethylhexanoate was soluble in dimethylformamide (DMF) up to at least 100 mg/mL. A maximum concentration of 5000 μg/plate was selected for Mutation Experiment 1, in order that initial treatments were performed up to this maximum recommended concentration according to current regulatory guidelines (OECD, 1997). A maximum concentration of 5000 μg/plate was also selected for Mutation Experiment 2. Test article stock solutions were prepared by formulating Isodecyl 3,5,5-trimethylhexanoate under subdued lighting in DMF with the aid of vortex mixing, to give the maximum required treatment concentration. The test article solutions were protected from light and used within approximately 4.5 hours of initial formulation.

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

other: histidine dependence, rfa character, uvrB character and resistance to ampicillin or ampicillin plus tetracycline

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Mutation Experiment 1 (S-9 +-) ; 5, 16, 50, 160, 500, 1600, 5000 µg/plate
Mutation Experiment 2 (S-9 +-) ; 160, 300, 625, 1250, 2500, 5000 µg/plate

Vehicle / solvent:

DMF

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, bacterial strain, test article concentration (where appropriate), positive and vehicle controls, absence or presence of S-9 mix.

Mutation Experiments
Isodecyl 3,5,5-trimethylhexanoate was tested for mutation (and toxicity) in five strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA102), in two separate experiments, at the concentrations detailed previously, using triplicate plates without and with S-9 for test article, vehicle and positive controls. These platings were achieved by the following sequence of additions to molten agar at 45±1°C:
• 0.1 mL bacterial culture
• 0.1 mL of test article solution/vehicle control or 0.05 mL of positive control
• 0.5 mL 10% S-9 mix or buffer solution
followed by rapid mixing and pouring on to Vogel-Bonner E agar plates. When set, the plates were inverted and incubated at 37±1°C protected from light for 3 days. Following incubation, these plates were examined for evidence of toxicity to the background lawn, and where possible revertant colonies were counted (see Colony Enumeration Section 4.4). As the results of Mutation Experiment 1 were negative, treatments in the presence of S-9 in Mutation Experiment 2 included a pre-incubation step. Quantities of test article, vehicle control solution (reduced to 0.05 mL) or positive control, bacteria and S-9 mix detailed above, were mixed together and incubated for 20 minutes at 37±1°C, with shaking, before the addition of 2 mL molten agar at 45±1°C. Plating of these treatments then proceeded as for the normal plate-incorporation procedure. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected in the assay. Volume additions for the Mutation Experiment 2 pre-incubation treatments were reduced to 0.05 mL due to the vehicle (DMF) employed in this study. This, and some other organic vehicles, are known to be near to toxic levels when added at volumes of 0.1 mL in this assay system when employing the pre-incubation methodology. By reducing the addition volume to 0.05 mL per plate, it was hoped to minimise or eliminate any toxic effects of the vehicle that may have otherwise occurred.

Toxicity Assessment
The background lawns of the plates were examined for signs of toxicity. Other evidence of toxicity may have included a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Colony Enumeration
Colonies were counted electronically using a Sorcerer Colony Counter (Perceptive Instruments) or manually where confounding factors such as contamination affected the accuracy of the automated counter.

Analysis of Results
Treatment of Data
Individual plate counts were recorded separately and the mean and standard deviation of the plate counts for each treatment were determined. Control counts were compared with the laboratory’s historical control ranges (Sections 7.3 and 7.4). Data were considered acceptable if the vehicle control counts fell within the calculated historical control ranges and the positive control plate counts were comparable with the historical control ranges. The presence or otherwise of a concentration response was checked by non-statistical analysis, up to limiting levels (for example toxicity, precipitation or 5000 µg/plate). However, adequate interpretation of biological relevance was of critical importance.

Acceptance Criteria
The assay was to be considered valid if all the following criteria were met:
1. The vehicle control counts fell within the laboratory’s historical control ranges as defined in Section 7.3
2. The positive control chemicals induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control confirming discrimination between different strains, and an active S-9 preparation.

Evaluation criteria:

Evaluation Criteria
For valid data, the test article was considered to be mutagenic if:
1. A concentration related increase in revertant numbers was ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
2. The positive trend/effects described above were reproducible.
The test article was considered positive in this assay if both of the above criteria were met.
The test article was considered negative in this assay if neither of the above criteria were met.
Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Biological relevance was taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

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:

not specified

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:

not specified

Vehicle controls validity:

valid

Untreated 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:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS
Toxicity, Solubility and Concentration Selection
Details of all treatment solution concentrations and final Isodecyl 3,5,5-trimethylhexanoate concentrations are provided in the Test Article Section 3.1.
Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl 3,5,5-trimethylhexanoate at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity, as would usually be manifest by a diminution of the background bacterial lawn and/or a marked reduction in revertant numbers, was observed.
Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160 - 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl 3,5,5-trimethylhexanoate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.
The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Data Acceptability and Validity
The individual mutagenicity plate counts were averaged to give mean values, which are presented in Section 8. From the data it can be seen that vehicle control counts fell within the laboratory’s historical ranges (Section 7.3). The positive control chemicals all induced increases in revertant numbers of ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle controls confirming discrimination between different strains, and an active S-9 preparation. The study therefore demonstrated correct strain and assay functioning and was accepted as valid.

Mutation
Following Isodecyl 3,5,5-trimethylhexanoate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 and TA100) or ≥3-fold (in strains TA1535 and TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Isodecyl 3,5,5-trimethylhexanoate mutagenic activity in this assay system.

Conclusions:

It was concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Executive summary:

SUMMARY

Isodecyl 3,5,5-trimethylhexanoate was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9), in two separate experiments.

All Isodecyl 3,5,5-trimethylhexanoate treatments in this study were performed using formulations prepared in dimethylformamide (DMF).

Mutation Experiment 1 treatments of all the tester strains were performed in the absence and in the presence of S-9, using final concentrations of Isodecyl 3,5,5 -trimethylhexanoate at 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, plus vehicle and positive controls. Following these treatments, no evidence of toxicity was observed, as would normally be manifest as a thinning of the background bacterial lawn or a marked reduction in revertant numbers.

Mutation Experiment 2 treatments of all the tester strains were performed in the absence and in the presence of S-9. The maximum test concentration of 5000 µg/plate was retained for all strains. Narrowed concentration intervals were employed covering the range 160 - 5000 µg/plate, in order to examine more closely those concentrations of Isodecyl 3,5,5-trimethylhexanoate approaching the maximum test concentration and considered therefore most likely to provide evidence of any mutagenic activity. In addition, all treatments in the presence of S-9 were further modified by the inclusion of a pre-incubation step. In this way, it was hoped to increase the range of mutagenic chemicals that could be detected using this assay system. Following these treatments, no evidence of toxicity was observed.

The test article was completely soluble in the aqueous assay system at all concentrations treated, in each of the experiments performed.

Vehicle and positive control treatments were included for all strains in both experiments. The mean numbers of revertant colonies fell within acceptable ranges for vehicle control treatments, and were elevated by positive control treatments.

Following Isodecyl 3,5,5-trimethylhexanoate treatments of all the test strains in the absence and presence of S-9, no increases in revertant numbers were observed that were ≥1.5-fold (in strain TA102), ≥2-fold (in strains TA98 or TA100) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This study was considered therefore to have provided no evidence of any Isodecyl 3,5,5 -trimethylhexanoate mutagenic activity in this assay system.

It was concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium when tested under the conditions of this study. These conditions included treatments at concentrations up to 5000 µg/plate (the maximum recommended concentration according to current regulatory guidelines), in the absence and in the presence of a rat liver metabolic activation system (S-9).

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 May 2017 - 07 November 2017

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:

21 July 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Range-Finder (+-); 15.63; 31.25; 62.50; 125.0; 250.0; 500.0;µg/mL
Mutation Experiment (+-); 7.813; 15.63; 31.25; 62.50; 125.0; 250.0; 500.0 µg/mL

Vehicle / solvent:

DMF diluted 100-fold in the treatment medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

Test System
The test system was suitably labelled to clearly identify the study number, test article (if required), test article concentration (if applicable), positive and vehicle control, presence and absence of S-9.

Cytotoxicity Range-Finder Experiment
Treatment of cell cultures for the cytotoxicity Range-Finder Experiment was as described below for the Mutation Experiment. However, single cultures only were used and positive controls were not included. The final treatment volume was 20 mL.
Following 3 hour treatment, cells were centrifuged (200 g), washed with tissue culture medium and resuspended in 20 mL RPMI 10.
Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival. The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air for 9 days. Wells containing viable clones were identified by eye using background illumination and counted.

Mutation Assay
Treatment of Cell Cultures
At least 10^7 cells in a volume of 17.8 mL of RPMI 5 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 0.2 mL vehicle, test
article, culture medium for the UTC or positive control solution was added. S-9 mix or 150 mM KCl was added as described (see deviation in Section 7.5). Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only
used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell
densities were determined using a Coulter counter and the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival as described.
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more than one unit may be responsible for an increase in mutant frequencies (Brusick, 1986; Scott et al., 1991). Osmolality and pH measurements on post-treatment media were
taken in the cytotoxicity Range-Finder Experiment.

Evaluation criteria:

Acceptance Criteria
The assay was considered valid if all of the following criteria were met:
1. The MF in the vehicle control cultures was considered acceptable for addition to the laboratory historical negative control database
2. The MF in the concurrent positive controls induced responses that were comparable with those generated in the historical positive control database and gave a clear, unequivocal increase in MF over the concurrent negative control
3. The test was performed with and without metabolic activation
4. Adequate numbers of cells and concentrations were analysable.

Evaluation Criteria
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Please see other information below

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

See section below

Toxicity

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations (125 to 500 μg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations discarded. The highest concentration analysed, (125 μg/mL) gave 298% and 98% RS, in the absence and presence of S-9 respectively (see following table).

Range-Finder Experiment - 3 Hour Treatment in the Absence and Presence of S-9

Concentration μg/mL	3 Hour Treatment –S-9 %RS	3 Hour Treatment +S-9 %RS
0	100	100
UTC	113	85
15.625	98	71
31.25	107	0†
62.5	142	0†
125 P,PP	298	98

†              No toxicity data available due to a possible technical error

UTC        Untreated control

%RS        Percent Relative Survival

P               Precipitation noted at time of treatment

PP             Precipitation noted at end of treatment incubation period

No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration analysed (125 μg/mL) as compared to the concurrent vehicle controls (measured data not reported).

In the Mutation Experiment seven concentrations, ranging from 7.813 to 500 μg/mL, were tested in the absence and presence of S-9. Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations (125 to 500 μg/mL) in the absence and presence of S-9. The lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentrations discarded. Seven days after treatment all remaining concentrations were selected to determine viability and 6TG resistance in the absence and presence of S-9. The highest concentration analysed (125 μg/mL) gave 97% and 94% RS in the absence and presence of S-9, respectively (see following table).

Mutation Experiment - 3 Hour treatment in the Absence and Presence of S-9

3 Hour Treatment –S-9			3 Hour Treatment +S-9
Concentration μg/mL	%RS	MF §	Concentration μg/mL	%RS	MF §
0	100	3.78	0	100	4.05
UTC	116	4.54	UTC	106	4.57
7.813	129	3.96 NS	7.813	103	3.05 NS
15.63	101	3.35 NS	15.63	94	4.95 NS
31.25	108	3.82 NS	31.25	85	4.69 NS
62.5	106	2.21 NS	62.5	90	5.44 NS
125 P PP	97	4.41 NS	125 P PP	94	3.89 NS
NQO 0.15	64	49.23	B[a]P 2	34	100.38
NQO 0.20	68	44.42	B[a]P 3	17	91.67

Linear trend tests on mutant frequency -S-9: Not significant (negative trend)

Linear trend tests on mutant frequency +S-9: Not significant

UTC       Untreated control

§              6TG resistant mutants/106 viable cells 7 days after treatment

%RS        Percent relative survival adjusted by post treatment cell counts

NS          Not significant

P             Precipitation noted at time of treatment

PP           Precipitation noted at end of treatment incubation period

Mutation

A summary of the results for the Mutation Experiment is shown in the previous table. The individual plate counts observed and the statistical analyses are shown in Table 8.1 to Table 8.16. The historical negative control ranges, based on the last 20 experiments performed in this laboratory, are presented in Section 7.4. The acceptance criteria were met and the study was accepted as valid.

When tested up to a precipitating concentration, no statistically significant increases in MF were observed following treatment with Isodecyl 3,5,5-trimethylhexanoate at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Conclusions:

It is concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

Executive summary:

Isodecyl 3,5,5-trimethylhexanoate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by a Mutation Experiment, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in dimethyl formamide (DMF).

A 3 hour treatment incubation period was used for each experiment.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 500 μg/mL (a precipitating concentration based on data previously generated in a solubility assessment). The highest concentration analysed was 125 μg/mL in the absence and presence of S-9 (limited by the appearance of post treatment precipitate), which gave 298% and 98% relative survival (RS), in the absence and presence of S-9, respectively.

In the Mutation Experiment seven concentrations, ranging from 7.813 to 500 μg/mL, were tested in the absence and presence of S-9. Seven days after treatment, the highest concentration analysed to determine viability and 6TG resistance was 125 μg/mL (limited by the appearance of post treatment precipitate), which gave 97% and 94% RS in the absence and presence of S-9, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

When tested up to precipitating concentrations, no statistically significant increases in MF were observed following treatment with Isodecyl 3,5,5-trimethylhexanoate at any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends indicating a clear negative result.

It is concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions employed.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 April 2017-31 October 2017

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)

Version / remarks:

23 July 2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Isodecyl 3,5,5-trimethylhexanoate, clients trade name of Wickenol 152 (CAS number 59231-35-5), batch number P7560, was a colourless liquid, with a molecular weight of 298.5. It was received on 10 April 2017 and stored at 15-25°C, protected from light. Purity was stated as UVCB -100% (broad mixture of isomers) and the expiry date was given as December 2018. The test article information provided by the Sponsor is considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Target gene:

Not applicable.

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human lymphocytes prepared form the pooled blood of two female doners

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian liver post-mitochondrial fraction (S-9)

Test concentrations with justification for top dose:

Range finder (+&-S9) 3+21 hour treatment: 1.814, 3.023, 5.039, 8.398, 14.00,23.33,38.88,64.80,108.0,180.0,300.0, 500.0 µg/mL
Range finder (-S9) 24+24 hour treatment:1.814, 3.023, 5.039, 8.398, 14.00,23.33,38.88,64.80,108.0,180.0,300.0, 500.0 µg/mL

Trial 1 (-S9) 3+21 Hour Treatments 5, 15, 30, 40, 45, 50, 55, 62.5, 65, 70, 80, 90,100, 150, MMC 0.2, MMC 0.3 µg/mL

Trial 1 (+S9) 3+21 Hour Treatments 10, 25, 50, 100, 150, 200, 350, 500, CPA 2, CPA 3 µg/mL

Trial 1 (-S9) 24+24 Hour Treatment 10, 20, 25, 30, 35, 40, 45, 62.5, 80, 100, VIN, 0.04, VIN, 0.06 µg/mL

Trial 2 (-S9) 24+24 Hour Treatments 5, 10, 15, 20, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 60, 80, 100, MMC, 0.20, MMC, 0.30, VIN, 0.04, VIN, 0.06 µg/mL

Trial 3(+S9) 3+21 Hour Treatments 25, 50, 100, 150, 200, 300, 400, 500, 600, 800, 1000, CPA, 2.00, CPA, 3.00 µg/mL

Vehicle / solvent:

DMF

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: vinblastine

Details on test system and experimental conditions:

Metabolic Activation System

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was obtained from Molecular Toxicology Incorporated, USA where it was prepared from male Sprague Dawley rats induced with Aroclor 1254. The S-9 was supplied as lyophilized S-9 mix (MutazymeTM), stored frozen at <-20°C, and thawed and reconstituted with purified water to provide a 10% S-9 mix just prior to use. Each batch was checked by the manufacturer for sterility, protein content, ability to convert
ethidium bromide and cyclophosphamide to bacterial mutagens, and cytochrome P-450-catalysed enzyme activities (alkoxyresorufin-O-dealkylase activities).
See Quality Control Statements for S-9. Treatments were carried out both in the absence and presence of S-9 by addition of either 150 mM KCl or 10% S-9 mix respectively. The final S-9 volume in the test system was 1% (v/v).

Ingredient Final Content per mL in: 10% S-9 mix
Sodium phosphate buffer pH 7.4 (SPB) 100 µmoles
Glucose-6-phosphate (disodium) (G-6-P) 5 µmoles
β-Nicotinamide adenine dinucleotide phosphate (NADP) (disodium) 4 µmoles
Magnesium chloride (MgCl2) 8 µmoles
Potassium chloride (KCl) 33 µmoles
Water To volume
S-9 100 µL

Blood Cultures

Blood from two healthy, non-smoking female volunteers from a panel of donors at Covance was used for each experiment as follows:

Experiment Donor Sex Donor Age (years) Donor Identity
Range-Finder Female 23, 27 10253, 8669
Micronucleus Experiment, Trial 1 Female 23, 27 10253, 8669
Micronucleus Experiment, Trial 2 Female 28, 33 8270, 8578
Micronucleus Experiment, Trial 3 Female 23, 27 10253, 8669

No donor was suspected of any virus infection or exposed to high levels of radiation or hazardous chemicals. All donors are non-smokers and are not heavy drinkers of alcohol. Donors were not taking any form of medication (contraceptive pill excluded). The measured cell cycle time of the donors used at Covance, Harrogate falls within the range 13±2 hours. For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 mL of pooled heparinised blood into 8.5 mL pre-warmed (in an incubator set to 37±1°C) HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide. Blood cultures were incubated at 37±1°C for approximately 48 hours and rocked continuously.

Test System

The test system was suitably labelled (using a colour-coded procedure) to clearlyidentify the study number, assay type, experiment number, treatment time, donor sex,test article concentration (if applicable), positive and vehicle controls, in the absence
and presence of S-9 mix.
4.2 Cytotoxicity Range-Finder
S-9 mix or KCl (1 mL per culture) was added appropriately.
Cultures were treated with the test article, vehicle or culture medium for the UTC(0.1 mL per culture). Positive control treatments were not included.The final culture volume was 10 mL. Cultures were incubated at 37±1°C for thedesignated exposure time.
4.3 Micronucleus Experiment
S-9 mix or KCl (1 mL per culture) was added appropriately.
Cultures were treated with the test article, vehicle, culture medium for the UTC orpositive controls (0.1 mL per culture).
The final culture volume was 10 mL. Cultures were incubated at 37±1°C for thedesignated exposure time.
This scheme is illustrated as follows:
Number of Cultures
Treatment S-9 Cytotoxicity Range-Finder Micronucleus Experiment
3+21* 24+24* 3+21* 24+24*
Vehicle control -
+
2
2
2 4
4
4
Untreated control -
+
1
1
1 2
2
2
Test article -
+
1
1
1 2
2
2
Positive controls -
+
2
2
2
* Hours treatment + hours recovery
For removal of the test article, cells were pelleted (approximately 300 g, 10 minutes),washed twice with sterile saline (pre-warmed in an incubator set to 37±1°C), andresuspended in fresh pre-warmed medium containing foetal calf serum and penicillin /streptomycin. Cyto-B (formulated in DMSO) was added to post wash-off culturemedium to give a final concentration of 6 µg/mL per culture.
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Duration ofTreatment(hours)
S-9 Hours after Culture Initiation*
Addition of Test
Article
Removal of Test
Article
Addition of
Cyto-B
Harvest Time
3 - 48 51 52** 72
24 - 48 72 73** 96
3 + 48 51 52** 72
* Approximate times
** Assuming approximately 1 hour for removal of test article at the post treatment wash-phase
Changes in osmolality of more than 50 mOsm/kg and fluctuations in pH of more thanone unit may be responsible for an increase in chromosome aberrations (Scott et al.,1991; Brusick, 1986). Osmolality and pH measurements on post-treatment incubation
medium were taken in the cytotoxicity Range-Finder Experiment.
4.4 Harvesting
At the defined sampling time, cultures were centrifuged at approximately 300 g for10 minutes, the supernatant removed and discarded and cells resuspended in 4 mL(hypotonic) 0.075 M KCl at 37±1°C for 4 minutes to allow cell swelling to occur.
Cells were then fixed by dropping the KCl suspension into fresh, coldmethanol/glacial acetic acid (7:1, v/v). The fixative was changed by centrifugation(approximately 300 g, 10 minutes) and resuspension. This procedure was repeated as
necessary (centrifuging at approximately 1250 g, 2-3 minutes) until the cell pellets were clean.
4.5 Slide Preparation
Lymphocytes were kept in fixative at 2-8°C prior to slide preparation for a minimumof 3 hours to ensure that cells were adequately fixed. Cells were centrifuged
(approximately 1250 g, two to three minutes) and resuspended in a minimal amount of fresh fixative (if required) to give a milky suspension. Several drops of cell
suspension were gently spread onto multiple clean, dry microscope slides. Slides wereair-dried then stored protected from light at room temperature prior to staining. Slideswere stained by immersion in 12.5 µg/mL Acridine Orange in phosphate buffered
saline (PBS), pH 6.8 for approximately 10 minutes and washed with PBS (withagitation) for a few seconds. The quality of the staining was checked. Slides were air-dried and stored protected from light at room temperature. Immediately prior toanalysis 1-2 drops of PBS were added to the slides before mounting with glasscoverslips.
4.6 Selection of Concentrations for the Micronucleus Experiment
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per
concentration. From these data the replication index (RI) was determined.
RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formulae as follows:
RI = number binucleate cells + 2 (number multinucleate cells)
total number of cells in treated cultures
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Relative RI (expressed in terms of percentage) for each treated culture was calculatedas follows:
Relative RI (%) = RI of treated cultures x100
RI of vehicle controls
Cytotoxicity (%) is expressed as (100 – Relative RI).
A selection of random fields was observed from enough treatments to determinewhether chemically induced cell cycle delay or cytotoxicity had occurred.
A suitable range of concentrations was selected for the Micronucleus Experimentbased on these toxicity data.
4.7 Selection of Concentrations for Micronucleus Analysis (MicronucleusExperiment Only)
Slides were examined, uncoded, for RI to a minimum of 500 cells per culture to determine whether chemically induced cell cycle delay or toxicity had occurred.
The highest concentration selected for micronucleus analysis following treatment in the presence of S-9 (Micronucleus Experiment, Trial 1) was the highest concentration tested (500 µg/mL). However, despite the observations of post treatment precipitate
within the range-finder treatment at concentrations of 500, 300 and 180 µg/mL, no concentration limiting precipitate was observed within Micronucleus Experiment,
Trial 1 +S-9 treatments.
A further +S-9 trial was conducted (Micronucleus Experiment , Trial 3) testing up to 1000 µg/mL and where post treatment precipitate was observed down to and including 500 µg/mL (with the precipitate observed floating at the top of the culture
tube rather than coating the cell pellet).
A maximum concentration of 500 µg/mL (the lowest precipitating concentration observed by eye at the end of the treatment period was selected for slide analysis (Aardema et al., 2011; OECD, 2016). Slides from precipitating cultures were checkedto confirm the presence of precipitate would not preclude analysis.It was considered possible that precipitate may have been present but missed from theMicronucleus Experiment, Trial 1 assessments. As such, both Trial 1 and Trial 3 +S-9
treatment data have been reported.
The highest concentrations selected for micronucleus analysis following treatments inthe absence of S-9 (50.00 µg/mL for 3 hour treatment and 32.50 µg/mL for the 24 hour treatment) were ones at which 50-60% cytotoxicity was achieved (OECD,
2016). Analysis of slides from highly toxic concentrations was avoided, where possible.
Slides from the highest selected concentration and at least two lower concentrations
were taken for microscopic analysis, such that a range of cytotoxicity from maximum
to little was covered (where applicable).
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The positive control concentrations analysed exhibited cytotoxicity values in the
range of 39% to 62% and in keeping with the approximate limits for the test article
concentration selection.
4.8 Slide Analysis
Scoring was carried out using fluorescence microscopy.
Binucleate cells were only included in the analysis if all of the following criteria were
met:
1. The cytoplasm remained essentially intact, and
2. The daughter nuclei were of approximately equal size.
A micronucleus was only recorded if it met the following criteria:
1. The micronucleus had the same staining characteristics and a similar morphology
to the main nuclei, and
2. Any micronucleus present was separate in the cytoplasm or only just touching a
main nucleus, and
3. Micronuclei were smooth edged and smaller than approximately one third the
diameter of the main nuclei.
For each treatment regime, two vehicle control cultures were analysed for
micronuclei. As the vehicle control data were considered acceptable, UTC were not
analysed.
Slides from the positive control treatments were checked to ensure that the system
was operating satisfactorily. One concentration from each positive control, which
gave satisfactory responses in terms of quality and quantity of binucleated cells and
numbers of micronuclei, was analysed. This pre-analysis slide check was conducted
under non-blinded conditions.
All slides for analysis were coded by an individual not connected with the scoring of
the slides, such that analysis was conducted under blind conditions. Labels with only
the study number, assay type, experiment number, the sex of the donor and the code
were used to cover treatment details on the slides.
One thousand binucleate cells from each culture (2000 per concentration) were
analysed for micronuclei. The number of cells containing micronuclei and the number
of micronuclei per cell on each slide was recorded. The microscope stage co-ordinates
of the first six micronucleated cells were recorded.
Nucleoplasmic bridges (NPBs) between nuclei in binucleate cells were recorded
during micronucleus analysis to provide an indication of chromosome rearrangement.
Various mechanisms may lead to NPB formation following DNA misrepair of strand
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breaks in DNA (Thomas et al., 2003). In this assay, binucleate cells with NPBs were
recorded as part of the micronucleus analysis.
Micronucleus analysis was not conducted on slides generated from the Range-Finder
treatments.
Slide analysis was performed by competent analysts trained in the applicable Covance
Laboratories standard operating procedures. The analysts were physically located
remote from the Covance facility, but were subject to Covance management and GLP
control systems (including QA inspection). All slides and raw data generated by the
remote analysts were returned to Covance Laboratories for archiving on completion
of analysis.

Evaluation criteria:

See section below.

Statistics:

Selection of Concentrations for the Micronucleus Experiment
Slides from the cytotoxicity Range-Finder Experiment were examined, uncoded, for proportions of mono-, bi- and multinucleate cells, to a minimum of 200 cells per
concentration. From these data the replication index (RI) was determined. RI, which indicates the relative number of nuclei compared to vehicle controls was
determined using the formulae as follows:
RI = [number binucleate cells + 2 (number multinucleate cells)] /[total number of cells in treated cultures]

Relative RI (expressed in terms of percentage) for each treated culture was calculated
as follows:
Relative RI (%) = [RI of treated cultures/RI of vehicle controls] x100

Cytotoxicity (%) is expressed as (100 – Relative RI).

A selection of random fields was observed from enough treatments to determine whether chemically induced cell cycle delay or cytotoxicity had occurred.

A suitable range of concentrations was selected for the Micronucleus Experiment based on these toxicity data.

Species / strain:

lymphocytes: Human

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

Additional information on results:

Micronucleus Analysis

Raw Data
The raw data for the observations on the test article plus positive and vehicle controls are retained by Covance Laboratories Ltd. A summary of the number of cells
containing micronuclei is given in Table 8.1 to Table 8.4.

Validity of Study
The data in Table 8.1 to Table 8.8, Section 7.3 and Text Table 4 to Text Table 8 confirm that:

1. The binomial dispersion test demonstrated acceptable heterogeneity (in terms of MNBN cell frequency) between replicate cultures (Table 8.5 to Table 8.8)
2. The frequency of MNBN cells in vehicle controls fell within the normal range (Section 7.3)
3. The positive control chemicals induced statistically significant increases in the proportion of MNBN cells. Both replicate cultures at the positive control concentration analysed under each treatment condition demonstrated MNBN cell frequencies that clearly exceeded the normal range (Table 8.1 to Table 8.4)
4. A minimum of 50% of cells had gone through at least one cell division (as measured by binucleate + multinucleate cell counts) in vehicle control cultures at the time of harvest (Text Table 4 to Text Table 8)
5. The maximum concentration analysed under each treatment condition met the criteria specified in Section 4.7.

Analysis of Data
Pulse (3 hour) treatment of cells with Isodecyl 3,5,5-trimethylhexanoate in the absence and presence of S-9 (both trials) resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed (Table 8.5, Table 8.6 and Table 8.8). The MNBN cell frequency of all Isodecyl 3,5,5-trimethylhexanoate treated cultures (all concentrations) fell within normal ranges (Text Table 4, Text Table 5, Text Table 8 and Section 7.3).

Extended 24 hour treatment of cells with Isodecyl 3,5,5-trimethylhexanoate in the absence of S-9 resulted in small but statistically significant increases in MNBN cells at the highest and an intermediate concentration analysed (20 and 32.5 µg/mg inducing 20% and 59% cytotoxicity respectively) (Table 8.3, Text Table 7 and Table 8.7). However, these increases were small and set against a very low concurrent vehicle control response (0.25% MNBN cells, versus a normal range of 0.1% to 0.9% MNBN cells) (Section 7.3). The group mean and all individual MNBN cell values (all concentrations) fell within normal ranges and despite a statistically significant linear trend, there was no real evidence of a true concentration related response. For concentrations analysed of 10, 20, 30 and 32.5 µg/mL (inducing 1%, 20%, 49% and 59% cytotoxicity respectively), the mean MNBN cell frequencies were 0.3%, 0.8%, 0.45% and 0.8% as compared to the normal range of 0.1% to 0.9% MNBN cells. As such, these weak statistical increases were considered of no biological importance. No test article related increases in cells with NPBs were observed (Section 7.3).

Conclusions:

It is concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9). Concentrations were analysed either up to 500 µg/mL (3+21 hour +S-9 treatment, concentration limited by post treatment precipitate), or to concentrations inducing 50-59% cytotoxicity (3+21 hour and 24+24 hour -S-9 treatments) in accordance with current regulatory guidelines for the in vitro micronucleus assay.

Executive summary:

Isodecyl 3,5,5-trimethylhexanoate was tested in an in vitro micronucleus assay using duplicate human lymphocyte cultures prepared from the pooled blood of two female donors in a single experiment. Treatments covering a broad range of concentrations, separated by narrow intervals, were performed both in the absence and presence of metabolic activation (S-9) from Aroclor 1254-induced rats. The test article was formulated in dimethylformamide (DMF) and the highest concentrations tested in the Micronucleus Experiment were determined following a preliminary cytotoxicity Range-Finder Experiment. Treatments were conducted (as detailed in the following summary table) 48 hours following mitogen stimulation by phytohaemagglutinin (PHA). The test article concentrations for micronucleus analysis were selected by evaluating the effect of Isodecyl 3,5,5-trimethylhexanoate on the replication index (RI). Micronuclei were analysed at three or four concentrations and a summary of the data is presented in the following table:

Treatment	Concentration (µg/mL)	Cytotoxicity (%)^$	Mean MNBN Cell Frequency (%)	Historical Control Range (%)^#	Statistical Significance
3+21 hour -S-9	Vehicle^a	-	0.55	0.20 to 1.00	-
MN Experiment	30	0	0.40		NS
Trial 1	45	22	0.25		NS
	50	50	0.30		NS
	*MMC, 0.20	39	5.10		p≤0.001
3+21 hour +S-9	Vehicle^a	-	0.65	0.20 to 1.07	-
MN Experiment	200	0	0.25		NS
Trial 1	350	0	0.35		NS
	500	0	0.60		NS
	*CPA, 2.00	62	2.65		p≤0.001
24+24 hour -S-9	Vehicle^a	-	0.25	0.10 to 0.90	-
MN Experiment	10	1	0.30		NS
Trial 2	20	20	0.80		p≤0.01
	30	49	0.45		NS
	32.5	59	0.80		p≤0.01
	*MMC, 0.20	59	49.65		p≤0.001
	*VIN, 0.04	54	7.05		p≤0.001
3+21 hour +S-9	Vehicle^a	-	0.65	0.20 to 1.07	-
MN Experiment	200	0	0.80		NS
Trial 3	400	2	0.55		NS
	500	3	0.60		NS
	*CPA, 3.00	40	3.25		p≤0.001
	^aVehicle control was DMF	$ Based on replication index		^#95th percentile of the observed range	NS Not significant
	* Positive control

A second trial of the 24+24 hour -S-9 treatment (Experiment 1, Trial 2) was conducted to meet acceptable toxicity limits for a maximum concentration for slide analysis.

No concentration limiting cytotoxicity or precipitate was observed following 3+21 hour +S-9 treatment. As such a second trial (Micronucleus Experiment, Trial 3) was conducted, increasing the concentration range. However, precipitate was observed but appeared at the top of the treatment cultures rather than settling out over the cell pellet restricting the maximum concentration to be analysed to 500 µg/mL. It was therefore likely that this occurred for the Experiment 1, Trial 1 treatment but the observation missed in error. Both sets of treatment data are therefore presented.

Appropriate negative (vehicle and untreated) control cultures were included in the test system under each treatment condition. The proportion of micronucleated binucleate (MNBN) cells in the vehicle cultures fell within current 95th percentile of the observed historical vehicle control (normal) ranges. It was therefore not considered necessary to analyse the untreated control cultures. Mitomycin C (MMC) and Vinblastine (VIN) were employed as clastogenic and aneugenic positive control chemicals respectively in the absence of rat liver S-9. Cyclophosphamide (CPA) was employed as a clastogenic positive control chemical in the presence of rat liver S-9. Cells receiving these were sampled in the Micronucleus Experiment at 24 hours (CPA, MMC) or 48 hours (VIN) after the start of treatment. All positive control compounds induced statistically significant increases in the proportion of cells with micronuclei. All acceptance criteria were considered met and the study was therefore accepted as valid. Pulse (3 hour) treatment of cells with Isodecyl 3,5,5-trimethylhexanoate in the absence and presence of S-9 (both trials) resulted in frequencies of MNBN cells which were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle controls for all concentrations analysed. The MNBN cell frequency of all Isodecyl 3,5,5-trimethylhexanoate treated cultures (all concentrations) fell within the 95th percentile of the current observed historical vehicle control (normal) ranges. Extended 24 hour treatment of cells with Isodecyl 3,5,5-trimethylhexanoate in the absence of S-9 resulted in small but statistically significant increases in MNBN cells at the highest and an intermediate concentration analysed (20 and 32.5 µg/mL inducing 20% and 59% cytotoxicity respectively). However, these increases were small and set against a very low concurrent vehicle control response (0.25% MNBN cells, versus a normal range of 0.1% to 0.9% MNBN cells). The group mean and all individual culture MNBN cell values (all concentrations) fell within normal ranges and despite a statistically significant linear trend, there was no real evidence of a true concentration related effect. As such, these weak statistical increases were considered of no biological importance. It is concluded that Isodecyl 3,5,5-trimethylhexanoate did not induce biologically relevant increases in micronuclei in cultured human peripheral blood lymphocytes following treatment in the absence and presence of an Aroclor-induced rat liver metabolic activation system (S-9). Concentrations were analysed either up to 500 µg/mL (3+21 hour +S-9 treatment, concentration limited by post treatment precipitate), or to concentrations inducing 50-59% cytotoxicity (3+21 hour and 24+24 hour -S-9 treatments) in accordance with current regulatory guidelines for the in vitro micronucleus assay.

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 registered substance did not induce mutations in any of the three studies presented. It was therefore considered that the registered substance failed did not meet the criteria for classification as a mutagen under the Classification, Labelling, and Packaging (CLP) regulation (1272/2008).