2,6-dimethylheptan-2-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Under the test conditions (GLP and OECD Guideline 471 without any deviation)., Dimetol is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains.

Under the test conditions, (GLP and OECD Guideline 487 without any deviation) Dimetol is not clastogenic or anuegenic in human lymphocytes with or without metabolic

activation.

In vitro Gene mutation endpoint is predicted to be negative with a high degree of confidence using the OECD Toolbox QSAR model. Dimetol falls within the applicability domain and the prediction has a strong degree of confidence p-value = 4.12E-03 (strong confidence).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

(Q)SAR

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE: QSAR Toolbox 3.4.0.17

2. MODEL (incl. version number) Database version: 3.8.8/3.1.2

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CAS 13254-34-7

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
QMRF attached which describes scientific validity of model

5. APPLICABILITY DOMAIN
QMRF attached which explains applicability of domain

6. ADEQUACY OF THE RESULT
The OECD Toolbox was used to predict mammalian cell gene mutation in DIMETOL.
The target chemical (Dimetol) FALLS within applicability domain
The prediction is based on 5 neighbours' values, 5 of them equal to prediction.
Prediction confidence is measured by the p-value = 4.12E-03 (strong confidence)

Guideline:

other: QSAR prediction

GLP compliance:

no

Remarks:

not relevant

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Conclusions:

in vitro Gene mutation endpoint is predicted to be negative with a high degree of confidence using the OECD Toolbox QSAR model.
Dimetol falls within the applicability domain and the prediction has a strong degree of confidence p-value = 4.12E-03 (strong confidence).

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 August - 09 December 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 In Vitro Mammalian Cell Micronucleus Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.49 In Vitro Mammalian Cell Micronucleus Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Name of test material (as cited in study report): Dimetol
- Chemical name (IUPAC): 2,6-dimethylheptan-2-ol
- Physical state: Clear colourless liquid
- Analytical purity: 99.6 %
- Lot/batch No.: PE00090447
- Production date: 10 March 2014
- Expiration date of the lot/batch: 28 May 2016
- Storage condition of test material: Stored at refrigerator (2-8 °C) protected from light

Target gene:

None

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

1.8 % (v/v) S9-fraction; S9 fraction prepared from male Sprague Dawley rats orally induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw)

Test concentrations with justification for top dose:

Dose range finding test: 17, 52, 164, 512 and 1442 μg/mL culture medium, 3 and 24 h in the absence of S9-mix and for 3 h in the presence of S9-mix

First cytogenetic assay:
Without and with S9-mix: 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time
Cytogenetic assay 1A:
With S9-mix: 150, 250, 350, 360, 370, 380, 390 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time
Cytogenetic assay 1B:
With S9-mix: 100, 350, 360, 370, 380, 390, 400, 410, 420 and 430 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Second cytogenetic assay:
Without S9-mix : 50, 100, 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 24 h exposure time, 24 h harvest time
Cytogenetic assay 2A:
Without S9-mix : 50, 100, 150, 175, 200, 225, 250 and 275 μg/mL culture medium; 24 h exposure time, 24 h harvest time

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: No correction was made for the purity/composition of the test substance. Dimetol was dissolved in DMSO of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany). Dimetol concentrations were used within 2 h after preparation. The final concentration of the solvent in the culture medium was 1.0 % (v/v).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

other: colchicine

Remarks:

Without metabolic activation: mitomycin C (0.25 and 0.38 μg/mL for a 3 h exposure period and 0.15 and 0.23 μg/mL for a 24 h exposure period); colchicine (0.1 μg/mL for a 3 h exposure period and 0.05 μg/mL for a 24 h exposure period)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation: cyclophosphamide (15 and 17.5 μg/mL for a 3 h exposure period)

Details on test system and experimental conditions:

PREPARATION OF CULTURE:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking, male volunteers (aged < 35 years). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2013) are presented below:
Dose range finding study: age 31, AGT = 13.5 h
First cytogenetic assay: age 34, AGT = 12.8 h; Cytogenetic assay 1A: age 31, AGT = 13.5 h; Cytogenetic assay 1B: age 22, AGT = 12.8 h
Second cytogenetic assay: age 22, AGT = 12.9 h; Cytogenetic assay 2A: age 31, AGT = 12.9 h

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.
Environmental conditions: Cultures were incubated in a humid atmosphere of 80 - 100 % (actual range 60 - 91 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.0 - 37.4 °C) for 46 ± 2 h and thereafter exposed to test item.

METHOD OF APPLICATION: in medium (RPMI 1640 medium)
- Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively) (Invitrogen Corporation) and 30 U/mL heparin (Sigma, Zwijndrecht, The Netherlands).

DURATION
- Exposure duration: Dose range finding test: 3 and 24 h (- S9-mix); 3 h (+ S9-mix); First cytogenetic assay: 3 h (± S9 mix); Second cytogenetic assay: 24 h (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): Dose range finding test: 24 h (± S9 mix); First cytogenetic assay: 27 h (± S9 mix); Second cytogenetic assay: 24 h (- S9 mix)
SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (during or after exposure of the test substance) the chemical cytochalasin B (5 μg/mL) was added to the cultures and incubated for 24 h.
STAIN (for cytogenetic assays): 5 % (v/v) Giemsa solution (in Sorensen buffer pH 6.8) for 10 - 30 min

NUMBER OF REPLICATIONS:
- Duplicate cultures per dose for test item, vehicle and positive controls

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of 500 cells per culture was counted, scoring cells with one, two or more nuclei (multinucleated cells).
- At least 1000 (with a maximum deviation of 5 %) binucleated cells per culture were examined by light microscopy for micronuclei. In addition, at least 1000 (with a maximum deviation of 5 %) mononucleated cells per culture were scored for micronuclei separately. Due to cytotoxicity the number of examined bi- or mononucleated cells in the positive control groups might be <1000.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity/cytostasis of test item in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index).
%Cytostasis = 100-100{(CBPIt – 1)/(CBPIc –1)}
CBPI = [(No. mononucleate cells) + (2 x No. binucleate cells) + (3 x No. multinucleate cells)] / [Total number of cells]
t = test substance or control treatment culture
c = vehicle control culture
Three analysable concentrations were scored for micronuclei. The number of micronuclei per cell was not recorded. The highest dose level examined for micronuclei were the cultures that produced 55 ± 5 % cytotoxicity. The lowest dose level had little or no cytotoxicity (approximately the same as solvent control). Also cultures treated with an intermediate dose level were examined.

OTHER:
The following criteria for scoring of binucleated cells were used:
- Main nuclei that were separate and of approximately equal size.
- Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
- Main nuclei that were linked by nucleoplasmic bridges.

The following cells were not scored:
- Trinucleated, quadranucleated, or multinucleated cells.
- Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).

The following criteria for scoring micronuclei were adapted from Fenech, 1996:
- The diameter of micronuclei should be less than one-third of the main nucleus.
- Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
- Micronuclei should have similar staining as the main nucleus.

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics.

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At a concentration of 1442 μg/mL Dimetol precipitated in the culture medium.

DOSE RANGE FINDING TEST:
- Without metabolic activation (- S9-mix); 3 h exposure time, 27 h harvest time: 0, 0, 7 and 11 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.
- Without metabolic activation (- S9-mix); 24 h exposure time, 24 h harvest time: 0, 9, 11 and 43 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.
- With metabolic activation (+ S9-mix); 3 h exposure time, 27 h harvest time: 0, 8, 5 and 8 % cytostasis was observed at concentrations of 0, 17, 52 and 164 μg/mL. At 512 and 1442 μg/mL, cell lysis was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. Historical control data generated from experiments performed between September 2010 and September 2013.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- In the first cytogenetic assay, Dimetol was tested up to 350 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence of S9- mix and up to 390 μg/mL for a 3 h exposure time with a 27 h harvest time presence of S9-fraction. Appropriate toxicity was reached at these dose levels.
The following dose levels were selected for scoring of micronuclei:
Without S9-mix: 150, 250 and 350 μg/mL culture medium (3 h exposure time, 27 h harvest time).
With S9-mix: 100, 360 and 390 μg/mL culture medium (3 h exposure time, 27 h harvest time).

- In the second cytogenetic assay, Dimetol was tested up to 150 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.
The following dose levels were selected for the scoring of micronuclei:
Without S9-mix: 50, 100 and 150 μg/mL culture medium (24 h exposure time, 24 h harvest time).

Remarks on result:

other: all strains/cell types tested

Table 7.6.1/1: Number of mononucleated or binucleated cells with micronuclei of human lymphocyte cultures treated with Dimetol in the first and second cytogenetic assay

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
First cytogenetic assay - without metabolic activation (- S9-mix); 3 h exposure time, 27 h harvest time
0	0	1	1	2	3	9	12
150	14	2	3	5	4	1	5
250	30	1	1	2	1	2	3
350	60	1	0	1	1	2	3
0.25 MMC-C	49	1	2	3	32	36	68***
0.1 Colch	91	19	22	41****	12	16	28**
First cytogenetic assay - with metabolic activation (+ S9-mix); 3 h exposure time, 27 h harvest time
0	0	0	0	0	3	4	7
100	10	2	0	2	2	1	3
360	33	1	0	1	5	5	10
390	53	3	1	4	5	6	11
15 CP	58	1	1	2	25	41	66***
Second cytogenetic assay - without metabolic activation (- S9-mix); 24 h exposure time, 24 h harvest time
0	0	1	0	1	1	1	2
50	20	0	1	1	2	3	5
100	40	0	0	0	2	1	3
150	61	1	1	2	3	1	4
0.15 MMC-C	50	1	1	2	17	19	36***
0.05 Colch	94	42	37	79***	1^2)	0^2)	1^2)

 

*) 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 (second cytogenetic assay);1000-1027 bi- and mononucleated cells were scored for the presence of micronuclei (first cytogenetic assay). 

Duplicate cultures are indicated by A and B.

2) 46 and 41 binucleated cells were scored in the A and B culture respectively.

Conclusions:

Under the test conditions, Dimetol is not clastogenic or aneugenic in human lymphocytes with and without metabolic activation.

Executive summary:

In an in vitro mammalian cell micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured human lymphocytes were exposed to Dimetol at the following concentrations:

Dose range finding test: 17, 52, 164, 512 and 1442 μg/mL culture medium, 3 and 24 h in the absence of S9-mix and for 3 h in the presence of S9-mix

First cytogenetic assay:

Without and with S9-mix: 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Cytogenetic assay 1A: With S9-mix: 150, 250, 350, 360, 370, 380, 390 and 400 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Cytogenetic assay 1B: With S9-mix: 100, 350, 360, 370, 380, 390, 400, 410, 420 and 430 μg/mL culture medium; 3 h exposure time, 27 h harvest time

Second cytogenetic assay:

Without S9-mix : 50, 100, 150, 200, 250, 300, 350 and 400 μg/mL culture medium; 24 h exposure time, 24 h harvest time

Cytogenetic assay 2A: Without S9-mix : 50, 100, 150, 175, 200, 225, 250 and 275 μg/mL culture medium; 24 h exposure time, 24 h harvest time

Prior to the mitosis (during or after exposure of the test substance) the chemical cytochalasin B (5 μg/mL) was added to the cultures and incubated for 24 h. After harvesting, the cells were then treated with a hypotonic solution, fixed, stained and examined for micronuclei.

In the first cytogenetic assay, Dimetol was tested up to 350 μg/mL for a 3 h exposure time with a 27 h harvest time in the absence of S9- mix and up to 390 μg/mL for a 3 h exposure time with a 27 h harvest time presence of S9-fraction. Appropriate toxicity was reached at these dose levels.

The following dose levels were selected for scoring of micronuclei:

Without S9-mix: 150, 250 and 350 μg/mL culture medium (3 h exposure time, 27 h harvest time).

With S9-mix: 100, 360 and 390 μg/mL culture medium (3 h exposure time, 27 h harvest time).

In the second cytogenetic assay, Dimetol was tested up to 150 μg/mL for a 24 h exposure time with a 24 h harvest time in the absence of S9-mix. Appropriate toxicity was reached at this dose level.

The following dose levels were selected for the scoring of micronuclei:

Without S9-mix: 50, 100 and 150 μg/mL culture medium (24 h exposure time, 24 h harvest time).

 

Test item 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 independently repeated experiments. The number of mono- and binucleated cells with micronuclei found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals produced a statistically significant increase in the number of binucleated cells with micronuclei (mitomycin C and cyclophosphamide) and mononucleated cells with micronuclei (colchicine) indicating the validity of the study.

 

Under the test conditions, Dimetol is not clastogenic or aneugenic in human lymphocytes with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 August - 19 September 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well conducted and well described study in accordance with GLP and OECD Guideline 471 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): Dimetol
- Physical state: Colourless liquid
- Analytical purity: 99.6 %
- Lot/batch No.: 9000473391
- Date of manufacture: 19 June 2002
- Expiration date of the lot/batch: 08 June 2004
- Stability under storage conditions: Stable
- Storage condition of test material: At room temperature in the dark
- Specific Gravity: 0.8151
- Stability in vehicle: Dimethyl sulfoxide: Not indicated

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

other: See table 7.6.1/1

Metabolic activation:

with and without

Metabolic activation system:

5 % (v/v) S9-fraction; S9 fraction prepared from liver homogenates of male Wistar rats intraperitoneally induced with a solution (20 % (w/v)) of Aroclor 1254 (500 mg/kg bw) in corn oil.

Test concentrations with justification for top dose:

Dose range finding test 1 (direct plate assay) and Dose range finding test 2 (preincubation assay):
- Dimetol was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

Mutation assay:
Direct plate assay:
- Tester strains TA1535, TA1537, TA98 and TA102: 10, 33, 100, 333, 1000 and 3330 µg/plate, in the absence and presence of S9-mix (doses were selected based on the results of the dose range finding test 1)

Preincubation assay:
- Tester strains TA1535, TA1537, TA98 and TA102: 3, 10, 33, 100, 333 and 1000 µg/plate, in the absence and presence of S9-mix (doses were selected based on the results of the dose range finding test 2)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Test substance preparation: The test substance was dissolved in DMSO of spectroscopic quality (Merck). Test substance concentrations were prepared directly prior to use and used within 4 h after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

See table 7.6.1/2

Positive control substance:

9-aminoacridine

sodium azide

cumene hydroperoxide

methylmethanesulfonate

other: 2-nitrofluorene, Daunomycin, 2-aminoanthracene, 1,8-dihydroxy anthraquinone

Remarks:

with and without metabolic activation

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: - All Salmonella typhimurium strains received from Dr. Bruce N. Ames, University of California at Berkeley, U.S.A.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

DURATION
- Preincubation period: 30 minutes at 37 °C, 70 rpm
- Incubation period: Plates were turned and incubated in the dark at 37 °C for 48 h in both direct plate and preincubation methods

NUMBER OF REPLICATIONS:
- 3 plates/dose for treatment, vehicle and positive controls

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of Dimetol, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

OTHER:
- Colony counting: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually.

Evaluation criteria:

No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

None

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation:
- Dose range finding test 1 (direct plate assay): The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards. Precipitation of Dimetol on the plates was observed at the start of the incubation period at the concentration of 5000 µg/plate and at the end of the incubation period at 3330 and 5000 µg/plate.
- Dose range finding test 1 (preincubation assay): The test substance did not precipitate in the top agar. Precipitation of Dimetol on the plates was not observed at the start or at the end of the incubation period in all tester strains.
- Mutation assay:
- Direct plate assay: Dimetol precipitated in the top agar at concentrations of 1000 and 3330 µg/plate. Precipitation of Dimetol on the plates was observed at the start and at the end of the incubation period at the concentration of 3330 µg/plate.
- Preincubation assay: Dimetol did not precipitate in the top agar. Precipitation of Dimetol on the plates was not observed at the start or at the end of the incubation period in all tester strains.

RANGE-FINDING/SCREENING STUDIES:
- The reduction of the bacterial background lawn and the reduction in the number of revertants is presented in Table 7.6.1/3, 7.6.1/4, 7.6.1/5 and 7.6.1/6

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for TA 1537 in the absence of S9-mix (preincubation assay; positive control). However, since this value was just outside the limit of the range and a more than three-fold increase was observed compared to the solvent control, the validity of the test was considered to be not affected.

Results of toxicity:

 

Table 7.6.1/3: Toxicity of Dimetol in the dose range finding test (direct plate test) – TA100

 

Without S9-mix			With S9-mix
Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
5000	moderate	extreme	3330	slight	No biologically relevant reduction in the number of revertant colonies
			5000	moderate	moderate

 

Table 7.6.1/4: Toxicity of Dimetol in the dose range finding test (preincubation test) – TA100

 

Without S9-mix			With S9-mix
Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
333	slight	moderate	333	slight	slight
1000	absent	complete	1000	extreme	microcolonies
3330	absent	complete	3330	absent	complete
5000	absent	complete	5000	absent	complete

 

Table 7.6.1/5: Toxicity of Dimetol in the direct plate test

 

Strains	Without S9-mix		With S9-mix
	Dose (µg/plate)	Revertant colonies	Dose (µg/plate)	Revertant colonies
TA1535	3330	slight	3330	extreme
TA1537	3330	No reduction in the number of revertant colonies	3330	No reduction in the number of revertant colonies
TA98	3330	slight	3330	slight
TA102	3330	slight	3330	slight

 

Table 7.6.1/6: Toxicity of Dimetol in the preincubation test

 

Strains	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial background lawn	Revertant colonies	Dose (µg/plate)	Bacterial background lawn	Revertant colonies
TA1535	333	moderate	No reduction in the number of revertant colonies	333	slight	No reduction in the number of revertant colonies.
	1000	extreme	microcolonies	1000	extreme	microcolonies
TA1537	333	slight	moderate	333	slight	No reduction in the number of revertant colonies.
	1000	extreme	microcolonies	1000	extreme	microcolonies
TA98	333	moderate	moderate	333	slight	No reduction in the number of revertant colonies.
	1000	extreme	microcolonies	1000	extreme	microcolonies
TA102	333	slight	moderate	333	No reduction of the bacterial background lawn	slight
	1000	extreme	microcolonies	1000	extreme	microcolonies

 

- All other concentrations, not mentioned here, showed no reduction in the bacterial background lawn or in the number of revertant colonies.

See the attached document for information on tables of results (mutation assay)

Conclusions:

Under the test conditions, Dimetol is not considered as mutagenic in S. typhimurium (TA1535, TA1537, TA98, TA100 and TA102) strains.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA1535, TA1537, TA98, TA100 and TA102) were exposed to Dimetol at the following concentrations:

Dose range finding test 1 (direct plate assay) and Dose range finding test 2 (preincubation assay):
- Dimetol was tested in tester strain TA100 with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix.

Mutation assay:

Direct plate assay:

- Tester strains TA1535, TA1537, TA98 and TA102: 10, 33, 100, 333, 1000 and 3330 µg/plate, in the absence and presence of S9-mix (doses were selected based on the results of the dose range finding test 1)

Preincubation assay:

- Tester strains TA1535, TA1537, TA98 and TA102: 3, 10, 33, 100, 333 and 1000 µg/plate, in the absence and presence of S9-mix (doses were selected based on the results of the dose range finding test 2)

 

Metabolic activation system used in this test 5 % (v/v) S9-fraction; S9 fraction prepared from liver homogenates of male Wistar rats intraperitoneally induced with a solution (20 % (w/v)) of Aroclor 1254 (500 mg/kg bw) in corn oil. Vehicle and positive control groups were also included in mutagenicity tests.

 

In the direct plate assay, Dimetol precipitated on the plates at dose levels of 3330 and 5000 µg/plate in TA100. Toxicity was observed at the dose level of 5000 µg/plate in the absence of S9-mix and at 3330 and 5000 µg/plate in the presence of S9-mix. Dimetol precipitated on the plates at 3330 µg/plate in the strains TA1535, TA1537, TA98 and TA102. Toxicity was observed in the tester strains TA1535 (with S9-mix), TA98 and TA102. In the preincubation assay, Dimetol did not precipitate in the top agar. Precipitation of Dimetol on the plates was not observed at the start or at the end of the incubation period in all tester strains. Toxicity was observed at dose levels of 333 µg/plate and upwards in TA100, TA1535, TA1537, TA98 and TA102 strains. Dimetol did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiments. The positive and vehicle controls induced the appropriate responses in the corresponding strains indicating the validity of the study.

 

Under the test conditions, Dimetol is not considered as mutagenic in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available