Reaction mass of 1-(2,6,6-trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one and 1-(2,6,6-trimethylcyclohex-1-en-1-yl)hepta-1,6-dien-3-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genemutations in bacterial cells (Ames) test according to OECD TG 471: Negative

Cytogenicity in mammalian cells, in vitro micronucleus according to OECD TG 487: Negative

Genemutation in mammalian cells (MLA) using read across from Galbascone (tested according to OECDTG 476): Negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 March, 2016 - 09 July, 2016

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:

adopted 26 September 2014

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
Average Generation Time (AGT): 12.9 - 13.0 h
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.

- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
With and without S9-mix, 3 hr exposure; 27 hr fixation: 5.4, 17, 52, 164 and 512 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 17, 52, 164, 512 and 1600 µg/mL
First cytogenetic test:
Without S9-mix, 3hr exposure; 27 hr fixation: 10, 50, 60, 65, 70 and 75 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 10, 75, 85, 95, 105 and 125 µg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 3hr exposure; 27 hr fixation: 10, 50 and 60 µg/mL
With S9-mix, 3hr exposure; 27 hr fixation: 10, 85 and 95 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 10, 20, 30, 40, 50, 70 and 110 µg/mL
The following dose levels were selected for scoring of micronuclei:
Without S9-mix, 24hr exposure; 24 hr fixation: 10, 40 and 50 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. the test substance was dissolved in dimethyl sulfoxide of spectroscopic quality. DMSO is accepted and approved by authorities and international guidelines.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: colchicine: 0.1 µg/mL for 3 h exposure period; 0.05 µg/mL for 24 h exposure period

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 512 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 164 µg/mL and above in the presence and absence of S9, 3 hr treatment/27 hr fixation and at dose levels of 52 µg/mL and above in the absence of S9 for the continuous treatment of 24 hr/24 hours fixation.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells:

Experiment 1:
Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 2 2 4 4 5 9
10 2 3 0 3 5 3 8
50 31 4 1 5 7 4 11
60 50 2 3 5 4 2 6
0.25 MMC-C 27 3 3 6 22 24 46***
0.1 Colch 80 22 30 52*** 3 2) 4 2) 7

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 1 2 3 5 4 9
10 8 0 2 2 3 5 8
85 39 2 2 4 4 6 10
95 52 1 0 1 5 2 7
15 CP 56 1 0 1 18 24 42***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) 421 and 402 binucleated cells were scored for the presence of micronuclei, respectively.

Experiment 2:

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time

Concentration (µg/ml) Cytostasis (%) Number of mononucleated cells with micronuclei 1) Number of binucleated cells with micronuclei 1)
1000 1000 2000 1000 1000 2000
A B A+B A B A+B
0 0 1 1 2 4 1 5
10 15 0 0 0 3 3 6
40 38 0 0 0 2 0 2
50 46 3 1 4 3 1 4
0.15 MMC-C 37 4 1 5 23 17 40***
0.05 Colch 95 32 44 76*** 2 2) 6 2) 8***

*) Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1) 1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2) 72 and 131 binucleated cells were scored for the presence of micronuclei, respectively.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:

Experiment 1:

Without metabolic activation (-S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 213 273 14 1.60
B 200 290 10 1.62
10 A 215 276 9 1.59
B 211 276 13 1.60
50 A 278 221 1 1.45
B 300 199 1 1.40
60 A 353 147 0 1.29
B 344 156 0 1.31
65 A 404 94 2 1.20
B 379 116 3 1.24
70 A 426 74 0 1.15
B 411 87 2 1.18
75 A 449 50 1 1.10
B 444 56 0 1.11
0.25 MMC-C A 286 212 2 1.43
B 272 228 0 1.46
0.38 MMC-C A 327 171 2 1.35
B 311 189 0 1.38
0.1 Colch A 432 63 5 1.15
B 454 44 2 1.10

With metabolic activation (+S9-mix)
3 hours exposure time, 27 hours harvest time

Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 170 311 19 1.70
B 190 307 13 1.65
10 A 199 284 17 1.64
B 209 276 15 1.61
75 A 244 251 5 1.52
B 246 251 3 1.51
85 A 298 199 3 1.41
B 292 208 0 1.42
95 A 350 150 0 1.30
B 331 167 2 1.34
105 A 398 101 1 1.21
B 401 99 0 1.20
125 A 466 34 0 1.07
B 472 28 0 1.06
15 CP A 341 158 1 1.32
B 361 139 0 1.28
17.5 CP A 399 101 0 1.20
B 379 121 0 1.24

Experiment 2:

Without metabolic activation (-S9-mix)
24 hours exposure time, 24 hours harvest time

Concentration µg/ml Culture Number of cells with ….nuclei CBPI
1 2 3 or more
0 A 115 337 48 1.87
B 125 326 49 1.85
10 A 182 290 29 1.69
B 150 319 31 1.76
20 A 177 306 17 1.68
B 177 308 18 1.68
30 A 202 288 10 1.62
B 190 296 14 1.65
40 A 238 261 1 1.53
B 236 261 3 1.53
50 A 286 212 2 1.43
B 254 244 2 1.50
70 A 449 53 0 1.11
B 417 85 0 1.17
110 A 479 21 0 1.04
B 500 8 0 1.02
0.15 MMC-C A 232 263 6 1.55
B 236 263 1 1.53
0.23 MMC-C A 320 179 1 1.36
B 292 209 1 1.42
0.05 Colch A 496 22 0 1.04
B 497 18 0 1.03

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mononucleated Binucleated
- S9-mix + S9-mix - S9-mix
3 hour exposure 24 hour exposure 3 hour exposure 3 hour exposure 24 hour exposure
Mean number of micronucleated cells
(per 1000 cells) 21.11 22.57 26.02 28.78 23.18
SD 28.25 27.75 12.96 25.69 15.59
n 210 204 108 210 204
Upper control limit
(95% control limits) 78.68 86.40 48.42 70.48 63.33
Lower control limit
(95% control limits) -36.45 -41.26 3.61 -12.92 -16.97

SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between January 2012 and June 2016.

- Negative (solvent/vehicle) historical control data:
Mononucleated Binucleated
+ S9-mix - S9-mix + S9-mix - S9-mix
3 hour exposure 3 hour exposure 24 hour exposure 3 hour exposure 3 hour exposure 24 hour exposure
Mean number of micronucleated cells
(per 1000 cells) 0.89 1.07 0.95 3.57 3.77 4.00
SD 0.92 1.10 1.27 2.55 2.48 2.62
n 102 104 99 102 104 99
Upper control limit
(95% control limits) 3.04 3.87 3.84 9.19 10.23 10.81
Lower control limit
(95% control limits) -1.25 -1.74 -1.95 -2.05 -2.68 -2.81

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between January 2012 and June 2016.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.
- Measurement of cytotoxicity used: The cytostasis / cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI).

Conclusions:

An in vitro micronucleus assay with the test substance was performed according to OECD 487 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Haxalon is not clastogenic or aneugenic in human lymphocytes.

Executive summary:

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the test substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 60 and 95 μg/mL for a 3 h exposure time with a 27 h fixation time in the absence and presence of S9 -mix, respectively. In the second cytogenetic assay, the substance was tested up to and including the cytotoxic concentration of 50 μg/mL for a 24 h exposure time with a 24 h fixation time in the absence of S9 -mix. Reliable positive and negative controls were included. the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. It is concluded that the test substance is not clastogenic or aneugenic in human lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May, 2016 - 25 May, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254.

Test concentrations with justification for top dose:

Direct plate:
- Dose range finding test:
TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Based on the results of experiment 1, the following dose levels were used:
- Experiment 1:
TA 1535, TA 1537 and TA 98 (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate
Preincubation:
- Experiment 2:
Based on the results of experiment 1, the following dose levels were used:
TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: Dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in dimethyl sulfoxide.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

100 μL/plate DMSO

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat): preincubation

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

Not performed.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 2: Slight to moderate reductions in the bacterial background lawn were observed in the absence and presence of S9.

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:

Experiment 1: Cytotoxicity, was observed in the presence of S9-mix at the highest concentration tested. Experiment 2: Slight to moderate reductions in the bacterial background lawn were observed in the absence and presence of S9.

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:

Experiment 1: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in the presence of S9-mix at the highest concentration tested.

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:

Experiment 2: Slight to moderate reductions in the bacterial background lawn was observed in the absence and presence of S9-mix.

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:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
Experiment 1: Precipitation of Hexalon on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period.
Experiment 2: Precipitation of Hexalon on the plates was observed at the start of the incubation period at concentrations of 512 μg/plate and upwards and at 5000 μg/plate at the end of the incubation period. In addition, precipitation at the end of the incubation period was observed at 1600 μg/plate in tester strain TA1535 in the absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES:
No toxicity was observed in the tester strains TA100 and WP2uvrA. No increase in the number of revertants was observed upon treatment with Hexalon under all conditions tested. In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the absence of S9-mix at the dose level of 5000 μg/plate. However, since the increase was not two-fold (a maximum of 1.2-fold was reached), this increase was not considered to be relevant.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98
S9-mix - + - + - +
Range 78 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907
Mean 785 228 653 387 1155 860
SD 167 105 290 143 370 323
n 1684 1662 1448 1536 1646 1686

TA100 WP2uvrA
S9-mix - + - +
Range 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 892 1404 1263 342
SD 178 327 461 165
n 1650 1677 1370 1410

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

- Negative (solvent/vehicle) historical control data:

TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424

SD = Standard deviation
n = Number of observations

Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1:
Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in tester strains TA1537 and TA98 in the presence of S9-mix at the highest concentration tested. In all other tester strains no toxicity was observed.
In tester strains TA1535, TA1537 and TA98 in the absence of S9-mix and in TA1537 in the presence of S9-mix, fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test item rather it is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.
Experiment 2:
In the second mutation assay, there were no biologically relevant decreases in the number of revertants. Slight to moderate reductions in the bacterial background lawn were observed in tester strains TA1535, TA1537 and TA100 in the absence and presence of S9-mix. In strains TA98 and WP2uvrA, no reductions in the bacterial background lawn were observed.
In tester strain TA1537, a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 512 μg/plate (presence of S9-mix). Since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item, rather it is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

Conclusions:

The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay performed according to OECD 471 (1997) and GLP principles.

Executive summary:

The mutagenic activity of the test substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate.

The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. In experiment 1, cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in tester strains TA1537 and TA98 in the presence of S9-mix at the highest concentration tested. In all other tester strains no toxicity was observed.

In the second mutation assay, there were no biologically relevant decreases in the number of revertants. Slight to moderate reductions in the bacterial background lawn were observed in tester strains TA1535, TA1537 and TA100 in the absence and presence of S9-mix. In strains TA98 and WP2uvrA, no reductions in the bacterial background lawn were observed.

The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read-across information from an analogue is used

Justification for type of information:

The gene mutation in mammalian cells information for Hexalon is derived from information of Galbascone on this endpoint. The read across justification is presented in the Genotoxicity Endpoint. The accompanying files are also attached there.

Reason / purpose for cross-reference:

read-across source

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

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: Test system L5178Y/TK+/-3.7.2C

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

There is an Ames test and an in vitro micronucleus test available for Hexalon, but not a mouse lymphoma assay. Therefore a read-across is performed to the close structural analogue Galbascone (CAS#56973 -85 -4) for this endpoint.

Ames test performed with Hexalon

The mutagenic activity of the test substance was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. In experiment 1, cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn, was observed in tester strains TA1537 and TA98 in the presence of S9-mix at the highest concentration tested. In all other tester strains no toxicity was observed. In the second mutation assay, there were no biologically relevant decreases in the number of revertants. Slight to moderate reductions in the bacterial background lawn were observed in tester strains TA1535, TA1537 and TA100 in the absence and presence of S9-mix. In strains TA98 and WP2uvrA, no reductions in the bacterial background lawn were observed. The substance did not induce a significant dose related increase in the number of revertant (His+) colonies in each of the four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and Escherichia coli reverse mutation assay.

In vitro micronucleus test performed with Hexalon

In an in vitro micronucleus assay, cultured peripheral human lymphocytes were exposed to different concentrations of the test substance (dissolved in DMSO), in the presence and absence of S9-mix according to OECD 487 guideline and GLP principles. In the first cytogenetic assay, the substance was tested up to and including cytotoxic concentrations of 60 and 95 μg/mL for a 3 h exposure time with a 27 h fixation time in the absence and presence of S9 -mix, respectively. In the second cytogenetic assay, the substance was tested up to and including the cytotoxic concentration of 50 μg/mL for a 24 h exposure time with a 24 h fixation time in the absence of S9 -mix. Reliable positive and negative controls were included. the test substance did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. It is concluded that the test substance is not clastogenic or aneugenic in human lymphocytes.

Mouse Lymphoma Assay with Galbascone

The mouse lymphoma assay with the substance was conducted according to OECD 490 guideline and GLP principles. In the first experiment, the substance was tested up to and including concentrations of 50 and 60 μg/mL in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 11 and 14% in the absence and presence of S9-mix, respectively. In the second experiment, the substance was tested up to a concentration of 60 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 20%. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses. In the absence of S9-mix, the substance did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modification in the duration of treatment. In the presence of S9-mix, the substance did not induce a significant increase in the mutation frequency. It is concluded that the substance is not mutagenic in the mouse lymphoma L5178Y test system.

Assessing the in vitro genemutation in mammalian cells of Hexalon (CAS #79-78-7) using Galbascone (CAS #56973-85-4)

Introduction and hypothesis for the analogue approach forin vitrogenemutation toxicity in mammalian cells.

Hexalon (CAS #79-78-7) has trimethyl-cyclohexene backbone with an alkyl chain to it. The alkyl chain has an alpha-beta conjugated ketone bond and has an allyl bond at the end of the chain. For this substance, an Ames test and in vitro micronucleus test are available. However, for Hexalon noin vitrogene mutation study in mammalian cells is available.

In accordance with Article 13 of REACH, lacking information can be generated by applying alternative methods such asin vitrotests, QSARs, grouping and read-across. In this case read across will be used for assessing thein vitrogenemutation toxicity in mammalian cells using Galbascone as an analogue. This analogue has a similar molecular structure compared to Hexalon and therefore information from Galbascone can be used to determine thein vitrogene mutations in mammalian cells of Hexalon (The molecular structures are presented in the data matrix).

Hypothesis:Hexalon (target) has similarin vitrogene mutation in mammalian cell effects compared to Galbascone (source) based on the similarity in their structures.

Available information:The genetic toxicity of Hexalon has been tested in an Ames test (according to OECD 471) and in anin vitromammalian micronucleus test (according to OECD 487). The genotoxicity of Galbascone has been tested in an Ames test (according to OECD 471), in anin vitrochromosome aberration study in mammalian cells (according to OECD 473), and in anin vitromammalian cell gene mutation test (according to OECD 490). All studies were performed in compliance with GLP and have therefore a reliability of 1. The studies performed with Hexalon and Galbascone were all negative for genotoxicity.

2. Target chemical and source chemical(s)

Chemical structures of the target and the source are shown in the data matrix.

3. Purity / Impurities

Hexalon contains one main constituent present at ca. 78%, a constituent present at ca. 10% and three additional constituents all present below 5%. Except one (1.3%) all minor constituents of Hexalon have similar structures: a ring with an alkyl chain with a ketone group. This minor one has more a ring type of structure instead of a chain which is not expected to have a more severe genotoxicity compared to the other constituents. Galbascone contains two constituents that are isomers.As a result it is not expected that the impurities of the source and target chemicals affect the read-across justification.

4. Analogue approach justification

According to Annex XI 1.5 read across can be used to replace testing when the similarity can be based on a common backbone and “common breakdown products via physical and biological processes, which result in structurally similar chemicals”. When using read across the result derived should be applicable for C&L and/or risk assessment and it should be presented with adequate and reliable documentation. This read across will be based on a common structural backbone and the formation of similar breakdown products. For this read across the ECHA guidance (2015, RAAF) is considered. Galbascone is selected as an analogue because among the fragrance group containing alpha-beta conjugated ketones, called ionones, Galbascone is the most similar to Hexalon because it also contains a double bond at the end of the alkyl chain which is generally not observed within other ionones (Belsito et al., 2007, 2013). For Galbascone in vitro genemutation in mammalian cells information is available.

Structural similarities and differences:Hexalon has a trimethyl-cyclohexene backbone with an alkyl chain attached to it. The alkyl chain has an alpha-beta conjugated ketone bond and has an allyl bond at the end of the chain. Structural similarities between Hexalon and Galbascone are the alkyl chain attached to the ring, having an allyl group at the end of this chain, and four C-atoms between this allyl group and the alpha-beta conjugated bond. Hexalon has this ketone bond outside the ring while Galbascone has it inside the ring.The functional group is the alpha-beta conjugated ketone group. At the end of the alkyl chain there is an allyl bond which is not considered to be very reactive as such. Galbascone has a somewhat similar backbone, similar functional group and also an allyl group at the end of the alkyl chain. The difference with Hexalon is that Galbascone has this alpha-beta conjugated inside the ring. This is expected not to influence the reactivity because both functional groups are slightly hindered by the ring structure: Galbascone having the group in the ring and Hexalon has this group just outside the ring.

Toxico-kinetic similarities and differences:To assess the genotoxicity at the port of entry the absorption via these routes is discussed.Absorption:Hexalon and Galbascone have similar molecular weight and physico-chemical properties indicating similar absorption characteristics (molecular weight (232 and 192 g/mol), the substances being liquids, log Kows (5.5 and 4.5), and vapour pressures (0.08 and 1.14 Pa at ca. 25 °C), respectively). The higher log Kow of Hexalon is still within the range of good oral absorption but it may have a somewhat lower dermal absorption. Via the inhalation route both substances will be also absorbed.Metabolism:Hexalon and Galbascone are anticipated to have similar metabolism on theoretical grounds. Possible primary and secondary steps of metabolism are presented in Belsito et al (2007) and Belsito et al, (2013). Primary metabolism may include reduction of the ketone group to a secondary alcohol; hydroxylation/oxygenation of the cyclohexene-ring; oxidation of the angular methyl groups; reduction of the double bonds in the exocyclic-alkenyl side chain to form dihydro derivatives. The double bond in the tail may be prone to epoxidation and subsequent reaction with epoxide hydrolases or glutathione transferase. These metabolites are likely to be conjugated before excretion. The difference is the one double bond in the ring of Hexalon non-conjugated, while in Galbascone it is conjugated. This extra double bond is not affecting the genotoxic potential as can be seen in the Ames, and the in vitro micronucleus. Also it does not change the genotoxic profile according to the OECD Toolbox.

Toxico-dynamic aspect: Reactivity:Hexalon and Galbascone both possess Michael addition reactivity (OECD Toolbox) based on alpha-beta conjugated ketone bond. The reactivity is considered similar in both substances. The reactive site of attack is on the alkyl side of the double bond. The alpha-beta conjugation in the ring possibly slightly diminishes this reactivity. Hexalon has the alpha-beta conjugation just outside the ring. The double bond is only one methyl group away from the ring. The ring actually may somewhat sterically hinder protein attack at the double bond position. Therefore reactivity of Hexalon and Galbascone at this site is considered similar. Both Hexalon and Galbascone also have a double bond at the end of the alkyl chain. The reactivity of the double bond at the end of the tail is also similar for both substances.

Experimental datasimilarity and difference:Hexalon and Galbascone are negative in Ames and in the in vitro micronucleus test (OECDTG 471 and 487) further supporting the analogy and the similar absence of in vitro genotoxicity effect.

Uncertainty of the prediction:There is no remaining uncertainty, in view of similarities in structure, toxico-kinetic (absorption and metabolism) and anticipated toxico-dynamic profile (reactivity) the read across is justified.

5. Data matrix

The relevant information on physico-chemical properties and toxicological characteristics are presented in the Data matrix in Table 1.

6. Conclusions for genemutations in mammalian cells

For Hexalon genotoxicity information is available from Ames (OECD 471) and of the in vitro micronucleus test (OECD 487) and not for gene mutations in mammalian cell. For the latter endpoint the information on gene mutations in mammalian cells of Galbascone is used using read across. Based on this Hexalon is negative for this endpoint.

Final conclusion on hazard, C&LPBT and risk characterization

Hexalon is negative for the genemutations in mammalian cells.

Based on the results from all available genotoxicity tests, classification of the substance is not warranted according to EU CLP (EC 1272/2008 and its updates).

Data matrix for the read across to Hexalon from Galbascone

Common names	Hexalon	Galbascone
Chemical structures*,
Chemical name	1-(2,6,6-Trimethylcyclohex-2-en-1-yl)hepta-1,6-dien-3-one	1-(5,5-dimethyl-1-cyclohexen-1-yl)pent-4-en-1-one
CAS no	79-78-7	56973-85-4
Einecs	201-225-9	260-486-7
REACH registration	2018	2018
Einecs	201-225-9	260-486-7
Smiles	O=C(C=CC(C(=CCC1)C)C1(C)C)CCC=C	CC1(C)CC(=CCC1)C(=O)CCC=C
Molecular weight	232	192
Appearance	Liquid	Liquid
Physico-chemical data
Melting point, °C	-20	-20oC
Vapour pressure, Pa	0.08	1.14
Water solubility, mg/l	79	89
Log Kow	5.5	4.5
Human health
Genotoxicity – Ames test	Negative (OECD 471)	Negative (OECD 471)
Genotoxicity – MLA	Read across	Negative (OECD 490)
Genotoxicity – in vitro micronucleus	Negative (OECD 487)	-
Genotoxicity – in vitro Chromosomal aberrations	-	Negative (OECD 473)

 

References:

D. Belsito, D. Bickers, M. Bruze, P. Calow, H. Greim, J.M. Hanifin , A.E. Rogers, J.H. Saurat, I.G. Sipes, H. Tagami., 2007. toxicologic and dermatologic assessment of ionones when used as fragrance ingredientsFood and Chemical Toxicology 45 (2007) S130–S167.

 

Belsito, D., Bickers, D., Bruze, M., Calow, P., Dagli, M.L., Fryer, A.D., Greim, H., Miyachi, Y., Saurat, J.H., Sipes, I.G., 2013, A toxicological and dermatological assessment of alkyl cyclic ketones when used as fragrance ingredients, Food and Chemical Toxicology 62, S1-S44.

Justification for classification or non-classification

Based on the negative results of the Ames test, the mouse lymphoma assay, and micronucleus assay, the test substance does not need to be classified for genotoxicity in vitro according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 and its updates.