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Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
1-Ethinylcyclohexanol did not reveal any indication for mutagenicity in the Ames test or in a gene mutation test in cultured mammalian cells (HGPRT assay). In an in vitro chromosomal aberration test in cultured V79 cells, positive effects were only noted in the presence of metabolic activation in a range where cytotoxic effects occurred. Thus, the aberrations are regarded as the result of disruption of cellular processes that would not be expected at lower doses, and considered as non-relevant to clastogenic risk at physiologically relevant concentrations. Furthermore, in a NMRI mouse bone marrow micronucleus assay 1-Ethinyl-1-cyclohexanol did not exert a cytotoxic effect in the bone marrow.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted in accordance with GLP.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 11 weeks
- Weight at study initiation: male mean value 36.0 g (SD +/- 1.7 g), female mean value 28.6 g (SD +/- 1.4 g)
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: single
Cage Type: Makrolon Type II/III, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
Bedding: granulated soft wood bedding (Rettenmaier & Söhne GmbH + Co. KG, 73494 Rosenberg, Germany)
- Diet: pelleted standard diet, ad libitum (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: minimum of five days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 45 - 65 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
- Vehicle used: corn oil
- Justification for choice of vehicle: relative non-toxicity for the animals
- Concentration of test material in vehicle:
Main experiment:
# Males: 187.5, 375 and 750 mg/kg b.w. as low, mid and high dose
# Females: 100, 200 and 400 mg/kg b.w. as low, mid and high dose
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.
- Lot/batch no.: MKBF8603V (Catalogue no.: C8267; Sigma-Aldrich Vertriebs GmbH, 82041 Deisenhofen, Germany)
Frequency of treatment:
Vehicle control, positive control and dose groups were treated once.
Post exposure period:
Samples of bone marrow were taken 24 hours after the treatment and for the highest dose level an additional sample was taken at 48 h after treatment.
Remarks:
Doses / Concentrations:
Males: 187.5, 375 and 750 mg/kg b.w. as low, mid and high dose
Basis:
actual ingested
based on the pre-experiment
Remarks:
Doses / Concentrations:
# Females: 100, 200 and 400 mg/kg b.w. as low, mid and high dose
Basis:
actual ingested
based on the pre-experiment
No. of animals per sex per dose:
six
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control: standard according to OECD TG 474
- Route of administration: orally
- Doses / concentrations: 40 mg/kg b.w.; dissolved in sterile water; volume administered: 10 mL/kg b.w



Tissues and cell types examined:
Bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The highest dose level represents in each gender the maximum tolerated dose as determined in the pre-experiment.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

DETAILS OF SLIDE PREPARATION:
The femora of sacrificed animals (using CO2 followed by bleeding) were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany) / Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Slides were evaluated using microscopes with 100x oil immersion objectives. Per animal (all animals per sex and test group) 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
See evaluation criteria above.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
other: vehicle control serves as negative control
Positive controls validity:
valid
Additional information on results:
On the basis of the pre-experiments 750 mg/kg b.w. for the males and 400 mg/kg b.w. for the females were estimated to be suitable as highest dose levels. Gender specific differences in toxicity were observed. In accordance with the guidelines both genders were used in the main experiment.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no - test item, positive control induced the appropriate response
- Ratio of PCE/NCE:
- Appropriateness of dose levels and route: yes, triggered by observed gender specific differences in toxicity
- Statistical evaluation: yes, non-parametric Mann-Whitney test
- All animals treated with the vehicle control (corn oil) did not express any clinical signs of toxicity.

Table 1: Rates of micronuclei (Males)

Test group

Dose
(mg/kg b.w.)

Sampling time (h)

PCEs with micronuclei
(%)

Range

PCE per 2000 erythrocytes

Vehicle control

          0

24

0.075

    1 -3

1292

Test item

   187.5

24

0.100

    0 -4

1294

Test item

      375

24

0.058

    0 -2

1307

Test item

      750

24

0.092

    0 -4

1276

Positive control

        40

24

2.075

  29 -54

1255

Test item

      750

48

0.142

    0 -8

1345

 

Table 2: Rates of micronuclei (Females)

Test group

Dose
(mg/kg b.w.)

Sampling time (h)

PCEs with micronuclei
(%)

Range

PCE per 2000 erythrocytes

Vehicle control

          0

24

0.108

    1 - 4

1295

Test item

      100

24

0.083

    0 - 3

1371

Test item

      200

24

0.175

    1 - 7

1381

Test item

      400

24

0.092

    0 - 4

1265

Positive control

        40

24

1.733

  18 -52

1260

Test item

      400

48

0.092

    1 - 3

1400

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In-vitro study: Bacterial systems

In a GLP-compliant reverse gene mutation assay according to OECD TG 471/472 (BASF AG, 1999), Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strain WP2 uvrA were exposed to 0, 20, 100, 500, 2500 and 5000 µg/plate 1-Ethinylcyclohexanol = 1-Ethynylcyclohexanol; (vehicle: DMSO) in a standard plate test in agar (SPT) or in a preincubation test (PIT). Plate assays were performed in triplicate in the presence and absence of mammalian metabolic activation (S9-mix) for each tester strain. Sterility controls were also included for each tester strain. An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system for every tester strain. Obtained data were within the range of the historical negative control data or sometimes decreased due to bacteriotoxicity observed at a concentration >= 2500 µg/plate in the preincubation test. The positive controls induced the appropriate responses in the corresponding strains and were like the negative vehicle controls within the range of the corresponding historical control data. The sterility controls showed no growth. No precipitation of the test substance was found. Thus, 1-Ethinylcyclohexanol was regarded as not mutagenic in the Ames test (Salmonella typhimurium/Escherichia coli reverse mutation assay) under the given experimental conditions. This study is classified as acceptable (key study).

 

In-vitro study: Mammalian cell gene mutation test

A GLP-compliant in vitro mammalian cell assay was performed according to OECD Guideline 476 (Harlan CCR, 2012). The test substance 1-Ethinyl-1-cyclohexanol (= 1-Ethynylcyclohexanol) was assessed for its potential to induce point mutations at the HPRT locus using V79 cells of the Chinese hamster in vitro.

The study was performed in two independent experiments, using identical procedures, each with and without liver microsomal activation. The test item was tested with the following concentrations:

Experiment I with and without S9 mix: 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (4 h exposure)

Experiment II without S9 mix: 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (24 h exposure)

Experiment II with S9 mix: 78.8, 157.5, 315.0, 630.0, 1260.0 µg/mL (4 h exposure)

According to the pre-experiment for toxicity the concentration ranges were selected to yield concentration related toxic effects.

Up to the highest investigated concentration no relevant and reproducible increase in mutant colony numbers was obtained in two independent experiments . The mutant frequency remained well within the historical range of solvent controls. No precipitation of the test substance was observed up to the maximum concentration of all experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion, it can be stated that in this mutagenicity assay and under the experimental conditions reported the test substance did not induce point mutations at the HPRT locus in V79 cells. The study is classified as acceptable key study).

 

In-vitro study: Chromosome aberration test

A GLP-compliant in vitro chromosome aberration test was performed according to OECD Guideline 473 and EU method B.10 (Harlan CCR, 2012). The test substance 1-Ethinyl-1-cyclohexanol (= 1-Ethynylcyclohexanol) was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese Hamster in vitro in the absence and presence of metabolic activation by S9 mix. Preparation of chromosomes was done 18 or 28 h after start of treatment with the test substance. In each experimental group, except the positive controls, four parallel cultures were used. Per culture 100 or 50 (some positive control) metaphases were scored for structural chromosomal aberrations.The following dose levels were evaluated with (S9) and without S9 (--) mix, the treatment period was 4 h (*), 18 h (**) or 28 h (***):

 

(--) 18 h preparation interval (Experiment I): 315.0, 630.0 and 1260.0 µg/mL (*)

(--) 18 h preparation interval (ExperimentIIA): 315.0, 630.0 and 1260.0 µg/mL (**)

(--) 18 h preparation interval (ExperimentIIB): 200.0, 400.0 and 500.0 µg/mL (**)

(--) 28 h preparation interval (ExperimentIIA): 157.5, 315.0 and 630.0 µg/mL (***)

(S9) 18 h preparation interval (ExperimentI): 315.0, 630.0 and 1260.0 µg/mL (*)

(S9) 28 h preparation interval (ExperimentIIA): 315.0, 630.0 and 1260.0 µg/mL (*)

(S9) 28 h preparation interval (ExperimentIIB): 200.0, 400.0, 1000.0, 1100.0 and 1260.0 µg/mL (*)

 

The concentration range of the test substance applied had been determined in a pre-experiment in which the test substance was applied up to a maximum concentration of 1260.0 µg/mL, as result no influence on solubility, pH value, or osmolarity was detected.

The cytogenetic evaluation of higher concentrations in the respective intervals (without S9 mix) was impossible due to strong test substance induced cytotoxic effects.

No relevant cytotoxicity, indicated by reduced mitotic indices and/or cell numbers could be observed in Experiment I in the absence and presence of S9 mix and in Experiment IIA in the presence of S9 mix.

However, in Experiment IIB in the presence of metabolic activation cell numbers were markedly reduced to 54.2 % of the solvent control at the highest evaluated concentration.

In the Experiment IIA in the presence of S9 mix two statistically significant values (5.3 and 4.8 % at 315.0 and 1260.0 µg/mL, respectively) were observed, which exceeded the laboratory’s historical solvent control data. In Experiment IIB this finding could be confirmed. A value of 8 % aberrant cells, excluding gaps was observed after treatment with 1260.0 µg/mL in a range where cytotoxic effects were observed.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item as compared to the rates of the solvent controls. No biologically relevant increase in endomitotic metaphases was noticed after treatment with the test item as compared to the control cultures.

Neither precipitation of the test substance in the culture medium nor a relevant influence on osmolarity or pH value was observed in the absence and presence of S9 mix.

Appropriate reference mutagens were used as positive controls and showed distinct increases of cells with structural chromosome aberrations.

1-Ethinyl-1-cyclohexanol was concluded to be positive in the presence of metabolic activation in the V79 cytogenetic assay in a range where cytotoxic effects occurred. Due to this, the aberrations may be the result of disruption of cellular processes that would not be expected at lower doses, and may therefore have no relevance to clastogenic risk at physiologically relevant concentrations The study is classified as acceptable (key study).

  

In-vivo study: Mammalian micronucleus assay

In a NMRI mouse bone marrow micronucleus assay (Harlan CCR, 2013), 6 animals/dose were treated orally once with 1-Ethinyl-1-cyclohexanol at doses of 0, 187.5, 375 and 750 mg/kg bw (males) or at doses of 100, 200 and 400 mg/kg bw (females) based on pre-investigations. The test substance was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. For micronuclei analysis bone marrow cells were harvested 24 hours after the treatment and for the highest dose level an additional sample was taken at 48 h after treatment. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that 1-Ethinyl-1-cyclohexanol did not exert a cytotoxic effect in the bone marrow. Clinical signs such as reduction of spontaneous activity, excitement, tumbling, hunchback, lack of alertness and/or ruffled fur were noted after treatment with the test substance. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. The positive control (40 mg/kg bw cyclophosphamide, administered orally) induced the appropriate response (substantial increase of induced micronucleus frequency). This GLP-conform study is classified as acceptable (key study). This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474, EU Method B.12 for in vivo cytogenetic mutagenicity data.


Justification for selection of genetic toxicity endpoint
The in vivo study on chromosomal aberration (mammalian micronucleus assay) was selected.

Justification for classification or non-classification

Based on the available results of the three different in vitro genetic toxicity studies and the outcome of an in vivo bone marrow micronucleus assay, 1-Ethinylcyclohexanol has not to be classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP).