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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

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

EC Number:

904-551-6

Molecular formula:

C16H24O

IUPAC Name:

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

Test material form:

liquid

Method

Species / strain

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.

Controls

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.

Results and discussion

Test results

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).

Applicant's summary and conclusion

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.