3,5,5-trimethylcyclohex-2-enone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Although in one standard mouse lymphoma assay a positive result was observed (Honma et al., 1999), the majority of in vitro genotoxicity studies revealed clearly negative results. Together with the negative in vivo results and the negative DNA binding assay (Thier et al, 1990), the overall conclusion is that isophorone is not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

study is part of a 13-week study (May 1979 - August 1979) and a 2-year carcinogenesis study (January 1980 - January 1982)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Principles of method if other than guideline:

Method: see: Haworth S, Lawlor T, Mortelmans K, Speck W and Zeiger E (1983).  Salmonella mutagenicity test results for 250 chemicals. Environ 
Mutagen.  5 (Suppl. 1), 3 -142.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci responsible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537

Details on mammalian cell type (if applicable):

Species/cell type: from Dr. B. Ames, Univ. of California

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat and hamster liver  fractions

Test concentrations with justification for top dose:

33, 100, 333, 1000, 3333 and 10000 (TA 1535), 100, 333, 1000, 3333, 10000 (TA 100), 33, 100, 333, 1000, 3333 (TA 98 and 1537) µg/plate

Vehicle / solvent:

water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Remarks:

potassium chloride

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: for details see below

Remarks:

no remarks

Details on test system and experimental conditions:

Type: Ames test
ADMINISTRATION: 
- Number of replicates: 3 per dose level, repeated
- Positive and negative control groups and treatment:    
sodium azide positive for TA 1535 and TA 100, 4-nitro-o-phenylenediamine positive for TA 98, 9-aminoacridine positive for TA 97 and TA 1537,  
2-aminoanthracene positive all strains, potassium chloride negative
- Solvent: H2O
- 3 investigations were performed: 1. without MA, 2. with MA (Arochlor-1254 liver rats) 3. with MA (Arochlor-1254 liver hamster)

Cells and test compound or solvent (water) were incubated for 20 minutes at 37 °C in the presence of either S9 or buffer (0.1 M PO4, pH 7.4).
After the addition of soft agar, the contents of each tube were poured onto 25 ml of minimal glucose bottom agar. When the top agar had solidified,
the plates were inverted and incubated at 37 °C for 48 hours (Haworth et al. 1983).  The analysis was performed twice, each in triplicate.

Evaluation criteria:

1. mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than
twofold
2. nonmutagenic response: when no increase in the number of revertants was elicited by the chemical
3. questionable response: when there was an absence of a clear-cut-dose-related increase in revertants; when the dose-related increases in the
number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity.

Statistics:

not reported

Species / strain:

other: Salmonella typhimurium TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: -S9: TA 100, 1535, 98 >=1000, TA 1537 >= 3333 µg/plate, +S9 (rat and hamster): only TA 1535 = 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: None (even at cytotoxic concentrations)
- Without metabolic activation: None (even at cyctotoxic concentrations)

CYTOTOXIC CONCENTRATION:
TA 98: >= 1000 µg/plate (-S9)
TA 100: >= 1000 µg/plate (-S9)
TA 1535: >= 1000 µg/plate (-S9); 1000 µg/plate (+S9)
TA 1537: >= 3333 µg/plate (-S9)

Remarks on result:

other: other: Salmonella typhimurium TA98, TA100, TA1535, TA1537

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, Isophorone was not mutagenic in strains TA 100, TA 1535, TA 1537, or TA 98 of Salmonella typhimurium in the
presence or absence of Aroclor 1254-induced Sprague-Dawley male rat or male Syrian hamster liver S9.

Executive summary:

The test substance Isophorone was tested for any mutagenic activity in the Ames Salmonella/microsomes test. Four histidine-

auxotrophic Salmonella typhimurium strains TA 98, TA 100, TA 1535, and TA1537 were treated with the test item by the pre-incubation method. Dose levels covering a total range of 33 to 10000 µg/plate, in triplicate both with and without the addition of a metabolising system (Aroclor induced rat and hamster liver S9 mix) were employed.

A mutagenic activity of Isophorone to any of the five tester strains was not observed with and without metabolic activation. It is therefore concluded, that Isophorone is not a bacterial mutagen.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-07-12 - 1988-08-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Principles of method if other than guideline:

Method: according to Ames et al. (1975), Mutat. Res. 31, 347-364

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Target gene:

mutated gene loci resposible for histidine auxotrophy

Species / strain / cell type:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 mix

Test concentrations with justification for top dose:

10-5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Remarks:

checked on strain TA 100 with aminoanthracene

Positive control substance:

other: 2-nitrofluorene; sodium azide; 9-aminoacridine

Remarks:

for details see below

Details on test system and experimental conditions:

Ames test
SYSTEM OF TESTING
- Metabolic activation system:  aroclor induced rat liver S9 mix; enzymatic activity tested with  aminoanthracene
ADMINISTRATION: 
- Number of replicates: 2
-  Application: solvent dimethyl sulfoxide
- Positive and negative control groups and treatment: 
2.5 µg nitrofluorene/plate: TA 98, TA 1538 (pos.)   
2.5 µg sodium azide/plate: TA 100, TA 1535 (pos.)   
50 µg aminoacridine/plate: TA 1537 (pos.)   
- negative control: no
- Pre-incubation: yes

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    
mutagenic effects (i.e  ratio of revertant rates treated/control >= 2)  at <= 5000 µg/plate with generally positive dose-response relationship in  any 
strain

Statistics:

not reported

Species / strain:

other: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- with metabolic activation: None
- without metabolic activation: None

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
negative

The test substance Isophorone proved to be non-mutagenic under the conditions of this study, both in the presence and in absence of Aroclor
induced liver microsomes for all the test strains even in addition of 5000 µg/plate and when the pre-incubation test was used.

Executive summary:

The test substance Isophorone was tested for any mutagenic activity in the Ames Salmonella/microsomes test. Five histidine-

auxotrophic Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA1537, and TA1538 were treated with the test item by the Ames test plate incorporation as well as the pre-incubation method. Dose levels covering a total range of 10 to 5000 µg/plate, in duplicate both with and without the addition of a metabolising system (Aroclor induced rat liver S9 mix) were employed.

A mutagenic activity of Isophorone to any of the five tester strains was not observed with and without metabolic activation. It is therefore concluded, that Isophorone is not a bacterial mutagen.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994-09 - 1995-03

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Microwell method

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus in mouse lymphoma cells

Species / strain / cell type:

other: Thymidine kinase (TK) locus of L5178Y tk+/- mouse lymphoma cells

Details on mammalian cell type (if applicable):

- cell type: L5178Y TK+/- mouse lymphoma cells Clone 3.7.2C
- source: from Dr. D. Clive (Glaxo Welcome Co., Research Triangle Park, NC)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from rat livers after phenobarbital- and  5,6-benzoflavone treatment

Test concentrations with justification for top dose:

no data (3 - 6 hr treatment)

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

positive with metabolic activation: cyclophosphamide

Migrated to IUCLID6: without metabolic activation

Details on test system and experimental conditions:

Mouse lymphoma assay
Isophorone was tested in two laboratories
Test: 3 test concentrations (2 replicates); 3-6 hr-treatment
Solvent: DMSO
Metabolic activation: rat livers after phenobarbital- and  5,6-benzoflavone treatment
Positive control (-S9): methylmethansulfonate (10 µg/ml)
Positive control (+S9): cyclophosphamide (3 µl/ml)

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
Following the statistical package analysis
- positive: when there were statistically significant responses (P< 0.05) in both the pair-wise comparison and the linear trend test
- equivocal: when the pair-wise comparison or linear trend test was significant (P< 0.05)
- negative: when no significant response was obtained in either procedure

Statistics:

All results were analyzed using a statistical package (Mutant TM, UKEMS, York, UK) in accordance with the UKEMS guidelines (Robinson et al., 1989).
This includes two procedures: one is pair-wise comparison of each treatment with the concurrent negative control and the other is testing for a linear
trend between mutation frequency and concentration.

Species / strain:

other: Thymidine kinase (TK) locus of L5178Y tk+/- mouse lymphoma cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

other: up to concentration: 10-20% relative survival (RS)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

- With metabolic activation: inconclusive (positive in one laboratory,  dose-dependent positive response * (max. Mutat. frequency < 2 times the  
spontanous one)) 
- Without metabolic activation: negative   * statistically significant

Remarks on result:

other: other: Thymidine kinase (TK) locus of L5178Y tk+/- mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
other: ambiguous with metabolic activation; negative without metabolic activation

The results indicate that, under the conditions of this mouse lymphoma assay, isophorone produced a negative response in absence of exogeneous
activation while in presence of exegenous activation an ambiguous result was observed (positive in one laboratory).

Executive summary:

Isophorone was tested in the L5178Y TK +/- mouse lymphoma mutagenesis assay (microwell method) in the presence and absence of

phenobarbital- and  5,6-benzoflavone induced liver S9-mix of Sprague-Dawley rats. Isophorone was tested in two laboratories and each experiment consisted of one solvent control, one positive control and at least three concentrations of the test chemical. All doses were carried out in duplicate. Cytotoxicity was was determined by relative survival (RS) following 3 -6 hr treatment.

The first laboratory obtained a dose-dependent and statistically significant positive response for isophorone with S9 mix, but the maximum mutation frequency was <2 times the spontaneous one. The second laboratory obtained negative responses at similar doses. In the absence of S9 mix, statistically significant positive responses were not obtained in either laboratory, although the second laboratory did not test isophorone at cytotoxic enough conditions.

Isophorone was toxic to the cultures up to concentrations of 10 - 20 % RS.

The results indicate that, under the conditions of this mouse lymphoma assay, isophorone produced a negative response in absence of exogeneous metabolic activation while in presence of exegenous metabolic activation an ambiguous result was observed (positive in one laboratory).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1984-03-05 to 1984-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Method: The experimental protocol is based on that described by Clive, D. and Spector, J.F.S. Laboratory procedure for assessing specific locus mutations at the TK locus in cultured L5178Y Mouse Lymphoma cells. Mutation Research 31: 17-29, 1975.

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus in mouse lymphoma cells

Species / strain / cell type:

other: L5178Y/TK +/-

Details on mammalian cell type (if applicable):

- cell type: L5178Y TK +/- mouse lymphoma cells Clone 3.7.2C
- source: from Dr. Donald Clive, Bourroughs Wellcome Company, North Carolina
- The cells were cryopreserved and stock cultures are prepared from reconstituted cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1242 and 1254 (2:1 mixture)  induced liver S9 mix of male Sprague-Dawley rats

Test concentrations with justification for top dose:

Concentration values are calculated:
119.6 - 1196 µg/ml (-S9);
81.9 - 818.8 µg/ml (+S9)
Remark: Original data are given in µl/ml: a density factor of 0.92 g/cm3 is assumed

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ethyl methanesulfonate (-S9); 7,12-dimethylbenz[a]anthracene (+S9)

Remarks:

for details see below

Details on test system and experimental conditions:

Mouse lymphoma assay
SYSTEM OF TESTING
- Metabolic activation system: Aroclor 1242 and 1254 (2:1 mixture)  induced liver S9 mix of male Sprague-Dawley rats
ADMINISTRATION: 
- Dosing: 16 dose levels decreasing approximately 100fold from 100 %  toxic to non-toxic; 
reported levels: 0.13; 0.18; 0.24; 0.32; 0.42, 0.56; 0.75; 1.0; 1.3 ul/ml (non-activated)  and 
0.067; 0.089; 0.12; 0.16; 0.21; 0.28; 0.38; 0.50; 0.67; 0.89 ul/ml (activated)
- Number of replicates: 1 culture (controls: 2); 3 counts
- Positive and negative control groups and treatment:    
ethyl methanesulfonate (-S9; 1.0 or 0.5 ul/ml: positive)   
7,12-dimethylbenz[a]anthracene (+ S9; 7.5 or 5.0 ug/ml: positive)   
DMSO only (negative)

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: 
- positive: positive dose response and >= 1 of 3 highest doses had a mutant  frequency 2-fold greater than background;
- equivocal: no dose response but any dose had a mutant frequency 2-fold  greater than background;
- negative: no dose response and no dose had a mutant frequency 2 -fold greater than background

Statistics:

Calculation programms: "Cell Culture Adjustment" and "Initial Toxicity" (TI 59 and Hewlett Packard PC)

Species / strain:

other: L5178Y/TK +/-

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: -S9: 1656 and 2208 µg/ml; +S9: 1104 and 1472 µg/ml produced 100 % lethality

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Concentration values are calculated: Original data are given in µl/ml; a  density factor of 0.92 g/cm3 is assumed.

Remarks on result:

other: other: L5178Y/TK +/-

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
negative

None of the cultures that were cloned, wether treated in the presence or absence of S-9, exhibites mutant frequencies which were significantly
greater than the mean mutant frequency of the solvent controls. The results indicate that, under the conditions of this test, Isophorone produced
a negative response in the presents and absence of exogenous metabolic activation.

Executive summary:

Isophorone was tested in the L5178Y TK +/- mouse lymphoma mutagenesis assay (MLA) in the presence and absence of Aroclor 1242 and 1254 (2:1 mixture) induced liver S9 mix of male Sprague-Dawley rats. The experiment was performed only once and all doses were tested in duplicate. In the presence of S9, isophorone concentrations of 0.13 to 1.3 ml/litre produced total growth of 12 % to 111 % compared to the control. In the presence of S9, isophorone concentrations of 0.067 to 0.89 ml/litre produced total growth of 9 % to 86 % compared to the control.

None of the cultures that were cloned, whether treated in the presence or absence of S9, exhibited mutant frequencies which were significantly greater than the mean mutant frequency of the solvent controls. The results indicate that, under the conditions of this test, isophorone produced a negative response in the presence and absence of exogenous metabolic activation.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

study is part of a 13-week study (May 1979 - August 1979) and a 2-year carcinogenesis study (January 1980 - January 1982), NTP-study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study

Principles of method if other than guideline:

Method: Galloway SM et al., Environ Mol. Mutagen. 10 (10), 1-175, with a few  modifications, see reference for details

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese Hamster Ovary (CHO) cells

Details on mammalian cell type (if applicable):

- stock cells were obtained from Litton Bionetics (Kensington, MD)
- all stock and experimental cultures were maintained at 37 °C in an atmosphere of 5% CO2 in air and 95% relative humidity

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from Aroclor 1254-induced male Sprague Dawley rats

Test concentrations with justification for top dose:

- S9: 50-1600 µg/ml, + S9: 250-1500 µg/ml

Vehicle / solvent:

Solvent: dimethyl sulfoxide (DMSO)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9 mix

Migrated to IUCLID6: 0.25 µg/ml; 1.00 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

+S9 mix

Migrated to IUCLID6: 15 µg/ml; 50 µg/ml

Details on test system and experimental conditions:

- Study design:
In the absence of S9 mix, CHO cells were incubated with test compound or solvent (DMSO) for 8-10 hours at 37 °C. Cells were then washed, and freshmedium containing colcemid (0.1 ug/ml) was added. After a further 2-3 hours of  incubation, cells were harvested by mitotic shake-off, fixed, and 
stained  in 6 % Giemsa.
In the presence of S9 mix, cells were incubated with test compound or solvent for 2 hours at 37 °C. Cells were then washed, medium  was added, and 
incubation continued for 8-10 hours. Colcemid (0.1 ug/ml)  was added for the last 2-3 hours of incubation; then cells were harvested  and fixed as above.  S9 mix was from the livers of Aroclor 1254-induced male Sprague-Dawley rats.
- Dosing
2 independent experiments:  
-S9 mix: 50, 160, 500, 1600 ug/ml   
-S9 mix: 250, 500, 1000, 1600 ug/ml   
+S9 mix: 250, 500, 1000 ug/ml   
+S9 mix: 750, 1000, 1250, 1500 ug/ml

- Analysis: 
100 or 200 cells were scored for each dose (cells with  chromosome number lower than 19 or higher than 23 were excluded)

Evaluation criteria:

The chromosome number wes recorded for each cell and chromosome or chromatid type aberrations were classified into three categories:
simple (breaks, fragments, double minutes), complex (interchanges, rearrangements), and other (pulverized, more than ten aberrations/cell).

Statistical analyses were conducted on both the slopes of the dose-response curves and the individual dose points. A statistically significant
(P< 0.003) trend test or a significantly elevated dose point (P< 0.05) was sufficient to indicate a chemical effect.

Statistics:

statistical method presented in Margolin et al. (1986)

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: >= 5000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

no data

Remarks on result:

other: other: Chinese Hamster Ovary (CHO) cells

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
other: negative with and without metabolic activation

Under the conditions of this test, isophorone did not induce chromosomal aberrations at concentrations up to 1600 (-S9) or 1500 (+S9) µg/ml,
respectively.

Executive summary:

Isophorone was tested in an in vitro cytogenetic assay (chromosomal aberrations, ABS) using Chinese hamster ovary (CHO) cell cultures. The test item was tested up to toxic doses both with and without exogenous metabolic activation. A liver fraction (S9) prepared from Arcolor 1254 -induced male Sprague-Dawley rats was used to provide exogenous metabolic activation. Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. Two seperate chromosomal aberration tests were conducted. The highest concentration tested was besed on solubility of the test substance.

Under the conditions of this test, isophorone did not induce chromosomal aberrations at concentrations up to 1600 (-S9) or 1500 (+S9) µg/ml, respectively.

Reference 6

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: according to Clive and Spector, Mutat. Res. 44, 269-278 (1975) and Clive et al., Mutat. Res. 59, 61-108 (1979)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase locus in mouse lymphoma cells

Species / strain / cell type:

other: L5178Y TK+/- cells

Details on mammalian cell type (if applicable):

- cell type: L5178Y TK +/- mouse lymphoma cells Clone 3.7.2C
- source: from Dr. Donald Clive, Bourroughs Wellcome Company, North Carolina
- stored in liquid nitrogen

Metabolic activation:

without

Test concentrations with justification for top dose:

50 - 1600 ug/ml (details see Test Conditions)

Vehicle / solvent:

DMSO (1%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

Mouse lymphoma assay (in the absence of S9)
Test procedure: Experiments were performed twice, and all doses were tested in duplicate,  except the solvent control (DMSO), which was tested in 
quintuplicate.  Cells (6 x 10e+5/ml) were treated for 4 hours at 37 °C in medium, washed,  resuspended in medium, and incubated for 48 hours at
37 °C. After  expression, 3 x10e+6 cells were plated in medium supplemented with  trifluorothymidine for selection of cells that were mutant at the  
thymidine kinase (TK) locus, and 600 cells were plated in nonselective  medium to determine the percentage of viable cells.

Trial 1: 0/50/100/200/400/800/1600 ug/ml
Trial 2: 0/400/600/800/1000/1200 ug/ml
Trial 3: 0/200/400/600/800/1000 ug/ml

Positive control: Ethylmethanesulfonate

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS: 
- positive: A test was considered positiv, when, out of three trials, a positive trial was reproducible
- negative: A test was considered negative, when, out of three trials, a positive response or a positive dose was not reproducible
- questionable: A test was considered questionable, when, out of three trials, neither a positive nor a negative response was reproduced

Statistics:

- mathematical model according to Lee and Caspary, 1983
- dose-trend test according to Barlow et al., 1972
- variance analysis of pair-wise comparisons of each dose against the vehicle control

Species / strain:

other: L5178Y TK+/- cells

Metabolic activation:

without

Genotoxicity:

other: positive; for details see below

Cytotoxicity / choice of top concentrations:

other: RTG reduced at 800, 1000 and 1200 µg/ml

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS: 
- Without metabolic activation: mutagenic Isophorone was toxic to the  cultures only at moderately high concentrations. Significant increases in 
 mutant fraction occurred in all three experiments, accompanied by a  reduction of relative total growth. 
The LOED (lowest effective dose) was 400 ug/ml in the first experiment,  where there was apparently no reduction in RTG (relative total growth)  
from the vehicle control level.  In the second experiment, only at 600 ug/ml there was evidence of  toxicity. The LOED in this experiment was 
800 ug/ml. However, the cloning  efficiency was low in this experiment (results questionable).  In the third experiment the 
LOED was 800 ug/ml.
CYTOTOXICITY: Relative total growth reduced at 800, 1000 and 1200 ug/ml

Remarks on result:

other: other: L5178Y TK+/- cells

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
positive

Isophorone was positive in the mouse lymphoma L5178Y/TK +/- assay in the absence of exogenous metabolic activation.

Executive summary:

Isophorone was tested in the L5178/TK +/- mouse lymphoma mutagenesis assay (MLA) in the absence of exogenous metabolic activation. The experiments were performed twice, and all doses were tested in duplicate, except the solvent control (DMSO), which was tested in quintuplicate.

Isophorone was toxic to the cultures only at moderately high concentrations. Significant increases in mutant fraction occurred in three experiments in the absence of S9 mix. The LOED was 400 µg/ml, where there was apparently no reduction in relative total growth (RTG). The RTG values at two lower concentrations, however, were considerably higher than in the vehicle control cultures. These anomalous results were due to higher suspension growth in the isophorone-treated cultures. In the second experiment, the lowest dose tested was 400 µg/ml, at which the RTG value was also higher than in the vehicle control cultures. Only at 600 µg/ml was there evidence of toxicity. The LOED in this experiment was 800 µg/ml. At this dose and two higher ones there was increases in mutant colony numbers as well as mutant fractions. This experiment was viewed with reservations, since the cloning efficiency was low. In a third experiment without S9, the LOED was again 800 µg/ml. The RTG was 24 % at this dose level.

The results indicate that, under the conditions of this test, isophorone produced a positive response in the absence of exogenous metabolic activation.

Reference 7

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: see "Details on test system and conditions"

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: Chinese Hamster lung fibroblast cells (CHL/IU)

Details on mammalian cell type (if applicable):

Cells were cultured in Eagle`s minimum essential medium supplemented with 10% heat-inactivated calf or fetal bovine serum. The doubling time of
the cells was around 15 h, and the modal chromosome number was 25.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix liver fraction from phenobarbital- and 5,6-benzoflavone pretreated male Sprague-Dawley rats

Test concentrations with justification for top dose:

standard procedure: 0.25-1.25 mg/ml; modified treatment: 0.75 - 1.75 mg/ml

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

-S9 mix (24 and 48 h)

Migrated to IUCLID6: 0.03 µg/ml

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

-S9 mix and +S9 mix (6h)

Migrated to IUCLID6: 10 µg/ml

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Metabolic activation system: Phenobarbital and 5,6-Benzoflavone  pretreated liver S9-fraction
- No. of metaphases analyzed: 100/dose
ADMINISTRATION: 
- Positive and negative control groups and treatment:    
negative: DMSO   
positive: mitomycin C, cyclophosphamide
Standard procedure: 
- without metabolic activation (24 h)
- without metabolic activation (48 h)
Modified treatment: 
Cells are treated for 6 h and then cultured in fresh  medium for another 18 h.
- without metabolic activation
- with metabolic activation

- CHROMOSOME ABERRATION TEST
Briefly, cells were treated with isophorone for 24 or 48 h continuously until cell harvest without an exogenous metabolic activation system
(standard procedure). Cells were also treated with isophorone for 6 h in the presence or absence of S9 mix. After 6 h treatments, cells were
cultured in fresh medium another 18 h (modified treatment). Colcemid (0.1 or 0.2 µg/ml) was added to the culture for the last 2 h. The cells were
incubated in 0.075 M KCL hypotonic solution for 15 min at 37°C and then fixed 3 times with ice-cold 1:3 acetic acid/methanol or ethanol. The
metaphase preparations were made by the conventional air-dry method with Giemsa staining.

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:    
positive: >= 10 % polyploidy or structural aberrations   
marginal: 5-10 %   
negative: < 5 %

Statistics:

no data

Species / strain:

other: Chinese Hamster lung fibroblast cells (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

other: standard procedure: -S9 (24 h): 1.25 mg/ml, -S9 (48 h): 1.0 mg/ml; modified treatment: -S9: > 1.25 mg/ml (not given), +S9: 1.75 mg/ml

Vehicle controls validity:

valid

Positive controls validity:

not specified

Additional information on results:

Standard treatment: Negative after 24 and 48 hours treatment
Modified treatment:
without S9: positive at 1.25 mg/ml (highest concentration tested)
with S9: positive at 1.5 mg/ml

Remarks on result:

other: other: Chinese Hamster lung fibroblast cells (CHL/IU)

Remarks:

Migrated from field 'Test system'.

no remarks

Conclusions:

Interpretation of results (migrated information):
ambiguous

This chromosomal aberration assay performed on Chinese hamster lung (CHL) cells was positive with isophorone with metabolic activation after a
modified treatment of the cells (cells are treated for 6 h and then cultured in fresh medium for another 18 h). Increased chromosome aberrations
without metabolic activation were only observed at cytotoxic concentrations.
After standard treatment (24 and 48 h treatment) without metabolic activation, isophorone did not induce a statistically significant increase in
chromosomal aberrations.

Executive summary:

The test substance isophorone was examined for its potential to induce structural chromosomal aberrations in Chinese hamster lung fibroblast cells (CHL/IU), in both the absence and presence of a metabolic activation system (S9 -mix). In this study the cells were treated with isophorone for 24 or 48 h continuously without an exogenous metabolic activation system (standard procedure). Cells were also treated with isophorone for 6 h in the presence and absence of S9-mix (liver S9 fraction prepared from phenobarbital- and 5,6 -benzoflavone-pretreated male Sprague-Dawley rats) (modified treatment). Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. In the standard procedure without metabolic activation, isophorone did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in the vehicle (DMSO) control cells. After modified treatment of the cells and with metabolic activation, isophorone was positive for Chinese hamster lung (CHL) cells. Increased chromosome aberrations without metabolic activation were only observed at cytotoxic concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

study conducted under the auspices of the National Toxicology Program (1983)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Method: NTP SLRL test method

GLP compliance:

not specified

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Strain:

other: Canton S and Basc stocks

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Age: adult
- Origin: Canton S and Basc stocks maintained at Brown University and the  University of Wisconsin

Route of administration:

oral: feed

Vehicle:

Solvent: ethanol

Details on exposure:

ADMINISTRATION: 
- Vehicle: ethanol, CAS RN 64-17-5 
- Solutions: two or three glass fiber discs were saturated with the test compound carried in solvent (ethanol) at the bottom of a standard glass vial.
Solutions were renewed at 24 hr and 48 hr.
- Duration of test: first mating after 72 hours of exposure
- Frequency of treatment: 
- Sampling times and number of samples: three broods, for each >= ca.  5000 chromosomes scored unless mutant frequency > 1.0 %
- Control groups and treatment: concurrent solvent (ethanol)

FOLLOW-UP TESTING:
2-3-day-old males were injected with 0.7 % NaCl solution containing the  test chemical at 12,500 ppm. At 24 hours postinjection, toxicity was tested
and survivors were mated.

Duration of treatment / exposure:

72 hours

Frequency of treatment:

1 time

Post exposure period:

no data

Remarks:

Doses / Concentrations:
2000 mg/kg
Basis:
nominal in diet

Control animals:

yes, concurrent vehicle

Positive control(s):

none

Tissues and cell types examined:

postmeiotic and meiotic germ cells

Details of tissue and slide preparation:

For the SLRL test, each male was mated individually to three Basc virgin females, then transferred to fresh Basc virgin females every 2 to 3 days to
make a total of three broods. To reduce the probability of recovering multiple lethals from one male, no more than 100 F1 females were mated over
the three broods from any P1 male. F2 cultures were scored as presumptive lethals if the number of wild-type males was 0, 1, or < 5% of the number
of Basc males. All putative lethals were confirmed through an additional generation.

Evaluation criteria:

Criteria for evaluating results:    
mutation frequency > 0.15 % (P < 0.05): positive   
mutation frequency > 0.10 % (P < 0.01): positive   
mutation frequency 0.10-0.15 % (P 0.01-0.05): equivocal   
other: negative

Statistics:

For the SLRL assay, a minimum of 5000 chromosomes was scored from each of the treated and concurrent control groups unless the mutant
frequency exceeded 1%.
The treated and control data were compared using a normal approximation to the binomial distribution. In addition, the trated data were compared to the historial control.

Sex:

male

Genotoxicity:

negative

Remarks:

no mutagenic effects were observed

Toxicity:

no effects

Remarks:

no toxic effects were observed

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

MORTALITY: 
- feeding: 15 %
- injection: 47 %
PERCENT STERILITY:
- feeding, injection: 0 %
PERCENT LETHALS:
- feeding:   0.11 %; control: 0.18 %
- injection: 0.22 %; control: 0.17 %

RESULT:  - feeding, injection: negative

no remarks

Conclusions:

Interpretation of results (migrated information): negative
In this Sex Linked Recessive Lethal (SLRL) test with Drosophila melanogaster no indication of a mutagenic effect was observed.

Executive summary:

In this Sex Recessive Lethal (SLRL) test with Drosophila melanogaster, fruit flies were fed a diet containing 2000 mg/kg isophorone. After 72 hrs of exposure, surviving males were mated. As feeding exposure was found to be non-mutagenic, 2 -3 day old males were injected with a 0.7 % NaCl solution containing 12,500 mg/l isophorone. 24 hrs post injection, males were mated. Again there was no indication of a mutagenic effect. Concurrent control males were treated with ethanol used to dissolve the test chemical.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study well documented, meets generally accepted scientific principles, acceptable for assessment

Principles of method if other than guideline:

Method: In-Vivo Micronucleus Test

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: CFLP

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST Animals: 
- Source: Anglia Laboratory Animals, Alconbury, Huntingdon
- Weight at study initiation: 19-23 g
- No. of animals per dose: 5 male, 5 female
- Housing: each group of 5 mice in a plastic disposable cage
- Diet: all animals were allowed free access to "Grain Harvester" Anglia Laboratory Animal Diet and tap water
- other: all animals were examined daily for signs of reaction and weighed prior to dosing
- Acclimation: following an acclimatization period of one week, they were randomly allocated to groups consisting of 5 male and 5 female mice
ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 21+/- 2°C
- Humidity, relative (%): 50 +/- 5%

Route of administration:

oral: gavage

Vehicle:

1 % methylcellulose

Details on exposure:

ADMINISTRATION: 
- Vehicle: isophorone was prepared as a suspension at concentrations of 450 - 1800 mg/kg bw (total) in 1 % methylcellulose using a Silverson high
speed mixer; all animals in all groups were dosed with the standard volume of 0.1 ml/10 g bw per dose.
- Duration of test: mice killed 6 hours after second dose
- Sampling times and number of samples: 6 h after dose # 2;
- Control groups and treatment:    
negative: vehicle   
positive: 14 mg/kg bw Mitomycin C (i.p. injection)

- number of animals in the positive control group: 5
- number of animals in the negative (vehicle) control group: 5

Duration of treatment / exposure:

2 equal exposures (half of total dose) separated by interval 24 hours

Frequency of treatment:

1 time

Post exposure period:

6 hours

Remarks:

Doses / Concentrations:
450; 900; 1800 mg/kg bw respectively (total)
Basis:
actual ingested

No. of animals per sex per dose:

test substance: 5
vehicle control: 5
positive control: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

 14 mg/kg bw Mitomycin C (i.p. injection)

Tissues and cell types examined:

polychromatic erythrocytes of the bone marrow from femur

Details of tissue and slide preparation:

Six hours after the second dose, the animals were killed by cervical dislocation, the femurs dissected out and bone marrow smears prepared.
The smears were fixed in methanol, defatted in xylene and stained (Giemsa). The stained smears were examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal.

Evaluation criteria:

not reported

Statistics:

not reported

Sex:

male/female

Genotoxicity:

negative

Remarks:

At all dosages of isophorone the group mean micronucleated cell count was comparable with the concurrent control value.

Toxicity:

yes

Remarks:

1800 mg/kg bw: period of lethargy

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

MORTALITY: 2 females and 1 male within 6 h after second dose in 1800  mg/kg bw group
CLINICAL SIGNS: 1800 mg/kg bw: period of lethargy
- at total dosages of 450 and 900 mg/kg bw, no toxic effects or mortalities were observed

MICRONUCLEATED CELL COUNT: 
neg. control:  mean  2.0; range 0-5
pos. control:  mean 82.5; range 68-107  
450 mg/kg bw: mean  2.2; range 0-5  
900 mg/kg bw: mean  1.9; range 0-6
1800 mg/kg bw: mean  2.1; range 1-4
- Six hours after the last treatment, the mean micronucleated cell counts and the bone marrow cytotoxicity were similar in all test groups and 
controls.

no remarks

Conclusions:

Interpretation of results (migrated information): negative Six hours after the last treatment, the mean micronucleated cell counts and the bone marrow cytotoxicity were similar in all test groups and controls.
From the results obtained it was concluded that isophorone did not exert any adverse effect on genetic material of male and female mice, when
administered orally, in this micronucleus test.

Executive summary:

In this micronucleus test for examined the incidence of isophorone of micronucleated polychromatic erythrocytes in mice, test substance doses of 450, 900 and 1800 mk/kg bw respectively were administered by oral gavage to CFLP mice of both sexes in two equal doses seperated by an interval of 24 hours. A negative control group was dosed in an identical manner with the vehicle, 1% methylcellulose. A positive control group was dosed by intraperitoneal injection with Mitomycin C, at a total dosage of 14 mg/kg bw. The mice were killed six hours after second dose and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse.

At all dosages of isophorone, the mean micronucleated cell counts and the bone marrow cytotoxicity were similar in all test groups and controls. Therefore, it was concluded from the results obtained that isophorone did not exert any adverse effect on genetic material, when administered orally, in this test procedure.

Reference 3

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

Study period:

1984-03-07 to 1984-09-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Method: Micronucleus Cytogenetic Assay

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS: 
- Age: 6-8 weeeks
- Source: Charles River Breeding Laboratories, Kingston, NY (USA)
- Weight at randomization: males 33-36 g, females 22-29 g
- No. of animals per dose: 5 males + 5 females per dose and sampling  time; 1 replacement animal per treated group
- Diet: ad libitum
- Water: ad libitum
- Housing: up to six animals per cage in plastic autoclavable cages with wire lids
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3 ° C
- Humidity (%): 50 +/- 20 %
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:

intraperitoneal

Vehicle:

corn-oil

Details on exposure:

ADMINISTRATION: 
- Vehicle: corn oil
- Control groups and treatment:   
negative: vehicle   
positive: 0.25 mg triethylenemelamine (TEM, CAS RN 51-18-3)/kg bw,  24  hours
- Frequency of treatment: single application
- Total volume applied: 10 ml/kg bw
- Duration of test: 12; 24; 48 hours
- Sampling times and number of samples: 12: 24; 48 h post dosing

Duration of treatment / exposure:

1 injection

Frequency of treatment:

1 time

Post exposure period:

12; 24; 48 hours

Remarks:

Doses / Concentrations:
498 mg/kg bw
Basis:
other: test dose was the LD20 of the test substance, determined in a preliminary toxicity study

No. of animals per sex per dose:

test substance: 5
vehicle control: 5
positive control: 5

Control animals:

yes, concurrent vehicle

Positive control(s):

0.25 mg triethylenemelamine (TEM, CAS RN 51-18-3)/kg bw,  24  hours

Tissues and cell types examined:

polychromatic erythrocytes of the bone marrow from femur

Details of tissue and slide preparation:

- Criteria for selection of M.T.D.: LD20
EXAMINATIONS: 
- Clinical observations: after dose administration
- Organs examined: femur bone marrow   
- Preparation: At sample collection, the femur was exposed and bone marrow aspirated into a syringe containing fetal calf serum. The cells was
washed, centrifuged, resuspended and smears prepared. May-Gruenwald-Giemsa stain was used to stain the bone marrow prparations.
- 1000 PCE (polychromatic erythrocytes) per animal were analysed for micronuclei; micronucleated normocytes were also counted

Evaluation criteria:

- Criteria for evaluating results: Statistically significant increase  (1-way analysis of variance and Duncan's multiple range test; P <= 0.05)  in 
micronucleated PCE, assessed considering reproducibility and sound science

Statistics:

t-test statistics

Sex:

male/female

Genotoxicity:

negative

Remarks:

No significant increase in the frequency of micronucleated polychromatic erythrocytes over the control was found with any group treated  with the test substance.

Toxicity:

yes

Remarks:

Heavy sedation immediately following test substance administration, no other clinical signs.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Main test: 3/18 dosed males and 3/18 dosed females died.
CLINICAL SIGNS: Heavy sedation immediately following test substance administration, no other clinical signs.

-see: " Remarks on results including tables"

MORTALITY: 
- Dose finding: Dead/total animals within 14 days
--------------------------------------------------------
  ml/kg bw    mg/kg bw    Males    Females    Total
--------------------------------------------------------
   0.00           0        0/5       0/5        0/10
   0.42         387        1/5       0/5        1/10
   0.46         424        0/5       0/5        0/10
   0.50         461        0/5       1/5        1/10
   0.52         479        0/5       1/5        1/10
   0.54         498        1/5       0/5        1/10
   0.56         516        2/5       2/5        4/10
   0.58         534        0/5       3/5        3/10
   0.60         553        2/5       3/5        5/10
   0.62         571        2/5       2/5        4/10
   0.64         590        3/5       4/5        7/10
--------------------------------------------------------
  LD20 = 0.54 +- 0.02 ml/kg bw
- Main test: 3/18 dosed males and 3/18 dosed females died.
CLINICAL SIGNS: Heavy sedation immediately following test substance  

administration, no other clinical signs.
EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: insignificant in all groups  

including positive control
GENOTOXIC EFFECTS: 
For the positive control a significant increase in the frequency of  

micronucleated polychromatic erythrocytes was observed. 

No significant  increase over the control was found with any group treated 

with the test  substance.
--------------------------------------------------------
Treatment     Sex   Time   Micron. PCE/1000PCE    % PCE
--------------------------------------------------------
Neg. contr.    m    12 h       0.6 +- 0.5           51
Neg. contr.    f    12 h       1.2 +- 1.3           51
Neg. contr.    m    24 h       0.8 +- 1.1           50
Neg. contr.    f    24 h       0.8 +- 0.8           51
Neg. contr.    m    48 h       0.8 +- 0.4           50
Neg. contr.    f    48 h       1.2 +- 1.1           51
Test subst.    m    12 h       1.4 +- 0.5           50
Test subst.    f    12 h       1.2 +- 1.3           51
Test subst.    m    24 h       0.4 +- 0.5           50
Test subst.    f    24 h       0.8 +- 1.1           50
Test subst.    m    48 h       1.4 +- 0.5           51
Test subst.    f    48 h       1.2 +- 1.3           52
Pos. contr.    m    24 h      37.6 +- 4.3 **        50
Pos. contr.    f    24 h      35.4 +- 3.7 **        52
--------------------------------------------------------
% PCE refers to total erythrocytes; * P<0.05; ** P<0.01

Conclusions:

Interpretation of results (migrated information): negative No significant increase in the frequency of micronucleated polychromatic erythrocytes over the control was found with any group treated  with the test substance.
The results of this study indicate that under the test conditions, isophorone did not induce micronucleated polychromatic erythrocytes in male and
female mice.

Executive summary:

In this in vivo mouse micronucleus assay, 498 mg/kg isophorone was administered i.p. to 5 male and 5 female CD-1 mice per group. This dose was selected as the maximum tolerated dose (= LD20 = MTD) based upon a preliminary toxicity study. Bone marrow polychromatic erythrocytes, collected 12, 24, and 48 hours after single treatment, were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control was found with any group treated  with the test substance. For the positive control (triethylene melamine, TEM) a significant increase in the frequency of PCE was observed.

Therefore, the conclusion is drawn, that isophorone is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female CD-1 mice.

Reference 4

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: other: DNA binding study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

DNA-binding study

GLP compliance:

not specified

Type of assay:

other: In order to clarify if direct inertaction of isophorone via covalent binding to DNA was responsible for the observed boderline effects in some mutagenicity assays, a DNA-binding study with F344 rats and B6C3F1 mice using labelled isophorone, was performe

Species:

other: rats and mice

Strain:

other: rat: Fischer 344; mouse: B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were placed individually, the mice in five groups of five randomized animals each, in closed all-glass metabolic cages; a continuous airstream was sucked through these cages and was subsequently drawn through soda lime, H2SO4 and H2O (for trapping of 14CO2 and water-soluble volatile metabolic products from the respiratory air). Food and water were administered ad libitum.

Route of administration:

oral: gavage

Vehicle:

neutral oil

Details on exposure:

Each animal of groups I - IV was dosed with a single dose of 500 mg unlabelled isophorone/kg body weight spiked with radiolabelled substance
(0.4 mCi per rat and 0.08 mCi per mouse; Group I: 25 female B6C3F1 mice; Group II: 25 male B6C3F1 mice; Group III: 5 female F344 rats; Group IV: 5 male F344 rats).
The animals received the radioactive isophorone preparation in neutral oil by gavage. To achieve a higher sensivity in group V (5 male F344 rats), this
group received the undiluted radioactive tracer (2 mCi; i.e., 0.4 mCi per rat) in neutral oil.

Duration of treatment / exposure:

single administration

Frequency of treatment:

1 time

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
500 mg/kg unlabelled isophorone spiked with  0.4 mCi per rat and 0.08 mCi per mouse
Basis:
nominal conc.

No. of animals per sex per dose:

rats: 5 animals
mice: 25 animals

Control animals:

other: only rats, undiluted radiactive tracer (2mCi; i.e., 0.4 mCi per rat) in neutral oil

Positive control(s):

none

Tissues and cell types examined:

isolated DNA from liver and kidney

Details of tissue and slide preparation:

Twenty-four hours after administration of the test compound, the animals were sacrificed and liver and kidneys were removed. For further
processing the organ samples were pooled from five mice each (groups I and II), whereas the organ samples of rats were processed individually
(groups III - IV). Cell nuclei from liver and kidneys were prepared by the standard method according to Rickwood and Birnie (1976) and the isolation
of DNA through hydroxyapatite chromatography was done as described by Bloom and Anderson (1978) with minor modifications. After removal of
remaining salts by dialysis and lyophilization, the DNA was dissolved in bidistilled water and precipitated with ethanol. The DNA samples were again
dissolved in bidistilled water and radioactivity of aliquots containing 100 - 1360 g DNA was measured by liquid scintillation counting.

Evaluation criteria:

negative result (none binding): values ranged within the 2 sigma margin (6dpm) of the background,
positive result (binding): values ranged above the 2 sigma margin (6 dpm) of the background

Statistics:

not reported

Sex:

male/female

Genotoxicity:

other: there was no binding of isophorone or its metabolites to DNA of liver and kidney

Toxicity:

not examined

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not examined

Additional information on results:

A significant attachment of radioactivity to DNA could not be found. All radioactivity values except one (B6C3F1 mice, No. 2, liver) ranged, within the 2sigma margin (6dpm) of the background, indicating that a significant attachment of radioactivity to the DNA could not be found.
It is concluded that neither isophorone nor its metabolite(s) are covalently bound to DNA. In addition, metabolically formed degradation products are also not incorporated into the DNA by de novo synthesis of DNA from labelled
fragments of the xenobiotic.

Table: Radioactivity associated with DNA/OF organs examined (means +/- SD; n = 5)

	Spec. Act*	Liver		Kidney
		µg DNA (counted	DNA	µg DNA (counted	DNA
Group I B6C3F1 mice 5 x 5 female	1110 µCi/mmol*	516 ± 251	11.1 ± 16.3	296 ± 86	4.4 ± 3.8
Group II B6C3F1 mice 5 x 5 male	820 µCi/mmol*	583 ± 260	10.3 ± 12.3	614 ± 176	6.1 ± 4.8
Group III F344 rats 5 female	570 µCi/mmol*	868 ± 289	2.4 ± 3.5	548 ± 64	2.3 ± 3.2
Group IV F344 rats 5 male	490 µCi/mmol*	1069 ± 220	1.7 ± 2.0	608 ± 154	1.6 ± 2.0
Group V F344 rats 5 male	52 mCi/mmol*	848 ± 487	1.3 ± 2.1	734 ± 143	0.0 ± 0.0

* Specific radioactivity after dilution with nonlabelled substance

Conclusions:

Interpretation of results (migrated information): other: no binding of isophorone or its metabolites to DNA of liver and kidney
Neither isophorone nor its metabolites are covalently bound to DNA. Degradation products are also not incorporated into newly formed DNA.
The carcinogenicity observed in the NTP study is probably not caused by genotoxic effects of isophorone.

Executive summary:

In this DNA binding study, male and female F344 rats (5 animals/group) and B6C3F1 mice (25 animals/group) were administered 500 mg/kg¹⁴C-isophorone. Livers and kidneys (target organs in the NTP carcinogenicity study) were removed after 24 hours. There was no binding of isophorone or its metabolites to DNA of these organs.

Therefore, it can be concluded, that the carcinogenicity observed in the NTP study is probably not caused by genotoxic effects of isophorone.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro Studies

Ames-tests

Several Ames tests with S.typhimurium TA 1535, TA 1537, TA 98 and TA 100 with and without S9 were negative (NTP, 1986; Hossack et al., 1978; Huels AG, 1988).

Mouse lymphoma tests

The mouse lymphoma tests were primarily negative. In the presence of metabolic activation only one positive result was obtained (Honma et al., 1999). Without metabolic activation some studies gave negative results (O´Donoghue et al., 1988; Honma et al., 1999), others positive results (McGregor at al., 1988). Positive results were found only at reduced RTG (relative total growth) values (McGregor et al., 1988). In one study, the incubation time was increased to 24 hours to render the test system especially sensitive for the detection of clastogens and spindle poisons. In this study, isophorone was positive in one of two trails (Honma et al., 1999).

Chromosomal aberration tests

In a cytogenetic assay with Chinese Hamster Ovary (CHO) cells, no significant increase in chromosomal aberrations was observed (Gulati et al., 1989; NTP, 1986). A further chromosomal aberration assay performed on Chinese hamster lung (CHL) cells was positive with isophorone with maetabolic activation only after a modified treatment of the cells (cells are treated for 6 hours and then cultured in fresh medium for another 18 hours). Increased chromosome aberrations without metabolic activation were only observed at cytotoxic concentrations (Matsuoka et al., 1996).

Sister chromatid exchange

A significant increase in SCE frequency at concentrations of 500 - 1000 mg/l induced by isophorone were only observed in the absence of S9 mix (no increase in the presence of Aroclor 1254 -induced rat liver S9 mix). As these high isophorone concentrations were cytostatic, increased SCE frequencies could only be detected after delayed harvest (Gulati et al., 1989).

Unscheduled DNA synthesis

Isophorone was tested for the induction of unschedulded DNA synthesis (UDS) in rat primary hepatocytes. No increase in the mean nuclear grain count (as compared to controls) or in the incidence of cells undergoing repair was detected at any dose level (O´Donoghue et al., 1988; Microbiological Associates, 1984).

In vivo Studies

Micronucleus assay

In a micronucleus assay, 498 mg/kg (= LD₂₀ = MTD) isophorone was administered i.p.to CD-1 mice (5 animals/sex). Significant increases in the number of micronucleated polychromatic erythrocytes (PCE) were not observed (O´Donoghue et al., 1988; Microbiological Associates, 1984).

In a second study with CFLP mice (5 animals/dose/sex; gavage doses of 450, 900, 1800 mg/kg given in 2 equal parts seperated by an interval of 24 hrs) a negative result was obtained 6 hours after the last dosage of isophorone (Hossack et al., 1978).

Sex Linked Recessive Lethal (SLRL) test

In a Sex Linked Recessive Lethal (SLRL) test with Drosophilia melanogaster, fuit lies were administered 2000 mg isophorone/kg and 12,500 mg isophorone/l, respectively. There was no indication of a mutagenic effect (Foureman et al., 1994).

DNA binding assay

In a DNA binding study, male and female F344 rats (5 animals/group) and B6C3F1 mice (25 animals/group) were administered 500 mg/kg¹⁴C-isophorone. Livers and kidneys (target organs in the NTP carcinogenicity study) were removed after 24 hours. There was no binding of isophorone or its metabolites to DNA of these organs (Thier et al., 1990).

It can be concluded, that the carcinogenicity observed in the NTP study is probably not caused by genotoxic effects of isophorone.

Justification for classification or non-classification

The majority of in vitro genotoxicity studies revealed clearly negative results. Together with the negative in vivo results and the negative DNA binding assay, the overall conclusion is that isophorone is not genotoxic and therefore must not be classified.