4-formyl-2-methoxyphenyl isobutyrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 May - 9 June 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

GLP compliance:

no

Remarks:

Conducted prior to development of GLP

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): Vanillin Isobutyrate

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

without

Metabolic activation system:

Rat liver homogenate metabolising system (S9Mix)

Test concentrations with justification for top dose:

The dose range of Vanillin Isobutyrate was 10 to 5000 µg/plate.

Vehicle / solvent:

Dimethyl sulphoxide was used as solvent.

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

no

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

Key result

Species / strain:

S. typhimurium, other: TA1535; TA1537; TA98; TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges.
Vanilline Isobutyrate caused a reduction in the growth of the bacterial lawn. The sensitivity of the bacterial strain to the toxicity of vanilline isobutyrate varied between strains and between exposure with or without S9Mix. No significant increase in the number of revertant colonies was recorded for any of the bacterial strains with any dose of Vaniline Isobutyrate either with or without metabolic activation.

Conclusions:

Vanilline Isobutyrate was found to be non-mutagenic under the test conditions.

Executive summary:

S. Typhimurium strains TA1535, TA1537, TA98, TA100 and E.Coli strains WP2uvra were treated with Vanilline Isobutyrate by the pre incubation method at six dose levels, in duplicate, both with and without the addition of a rat liver homogenate metabolising system (S9 Mix). In this case, the dose range of the Vanilline Isobutyrate was 10 to 5000 µg/plate. The method used conforms with the OECD 471 and 472 guideline.

All positive control chemicals gave increases in revertants, both with and without the metabolising system, within expected ranges. Vanilline Isobutyrate caused a reduction in the growth of the bacterial lawn. The sensitivity of the bacterial strain to the toxicity of vanilline isobutyrate varied between strains and between exposure with or without S9Mix. No significant increase in the number of revertant colonies was recorded for any of the bacterial strains with any dose of Vaniline Isobutyrate either with or without metabolic activation. Vanilline Isobutyrate was found to be non-mutagenic under the test conditions.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

strains not included (i.e. E. coli or S. typhimurium TA102)

GLP compliance:

yes

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): Isobutavan Y-09607 (13.12.1982)
- Physical state: thick yellow liquid
- Received at laboratory on December 21, 1982

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsome fraction of Aroclor-induced rats for metabolic activation

Test concentrations with justification for top dose:

Preliminary testing to assess the chemical toxicity of the test substance for the bacteria was performed on 0, 5 µg, 50 µg, 500 µg, 5 mg and 50 mg. Due to the results showing that 5 mg of test substance per plate was slighly toxic whereas 500 µg not showing any effect, the dose of 1500 µg/plate was chosen as the highest dose level for the mutagenicity.

Main test concentrations: 0, 0.19, 0.56, 1.67, 5.00 and 15.00 mg/mL in water

Vehicle / solvent:

The test substance was dissolved in DMSO prior to use, dosing solutions were made up with water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: hycanthone methanesulphonate, 2-aminoanthracene

Details on test system and experimental conditions:

The procedure is as follows:
Molten top agar (2 mL at 26°C) was added in this sequence: 0.1 mL of grown culture of one of the tester strains, 0.1 m of the appropriate dilution/suspension of the test product and 0.5 mL of the S-9 mix, if any. The ingredients were throughly mixed and the mix was poured onto minimal glucose agar plates.

After the top agar has set, the plates were incubated at 37°C for three days. The colonies (revertants which are histidine independent) were counted and the background lawn of bacterial growth was examined microscopically. Four to five different doses of the test product as well as a solvent control and a positive control were tested with each 5 different strains (TA 1535,1537,1538,98,100) with and without addition of the liver microsomes activation system. All determinations were made in triplicate.

S-9, S-9 mix and the test product were checked for sterility. Positive controls include direct as well as indirect mutagens. The dose range used in the mutagenesis assay is based on the results of the preliminary test performed with one of the bacterial strains without S-9 mix, using one plate per dose level-to assess the toxicity of the compound for the bacteria. Therefore, 1.5 mg per plate was chosen as the highest dose level was used.

Petri plates containing minimal glucose agar and an overlay of soft agar with bacteria and S-9 mix, if any, but without test substance, were prepared as per above paragraph for the plate incorporation assay. Within one hour after preparation and settings of the soft agar overlay, the plates were placed without lids in desiccators.

After an appropriate exposure period at 37°C, the various test atomspheres were removed. The petri plates were closed and removed from the dessiccators and again incubated at 37°C until a total incubation period of 3 days have been reached. Then the bacterial colonies were counted and the background lawn of bacterial growth examined. Concentrations and incubation times used in the test where chosen on the basis of the results in a pilot investigation

Evaluation criteria:

A positive response in the assay is taken to be a two fold or greater increase in the mean number of revertant colonies appearing in the test plates over and above the background spontaneous reversion rate observed with the solvent, together with evidence of a dose response.

Species / strain:

S. typhimurium, other: TA 1535; TA 1537; TA 98; TA 100; TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Exposure to the test substance did not increase the numbers of the Histidine revertants with any of the five tester strains, either in the absence or in the presence of the S-9 mix.
Within the range of concentrations tested, no indications of toxicity were obtained;the background lawn of bacterial growth in control and test plates were comparable.

Sample	Dose µg/plate		TA 1535		TA 1537		TA 1538		TA 98		TA 100
			-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Isobutavan	0.00	Mean:        CV:	33	20	10	14	15	32	33	45	114	114
			53	24	12	18	16	33	36	45	151	120
			55	24	13	25	27	36	43	68	159	133
			47	23	12	19	19	34	37	53	141	122
			26	10	13	29	34	6	14	25	17	8
Isobutavan	18.52	Mean:   CV:	40	15	7	9	8	28	25	36	105	132
			45	19	15	15	14	28	26	41	116	132
			48	19	16	16	14	48	33	52	180	151
			44	18	13	13	12	35	28	43	134	138
			9	13	39	28	29	33	16	19	30	8
Isobutavan	55.56	Mean:        CV:	37	14	3	15	13	28	28	44	117	120
			50	17	6	21	13	37	29	51	162	136
			60	25	14	24	15	42	39	51	175	147
			49	19	8	20	14	36	32	49	151	134
			24	30	74	23	8	20	19	8	20	10
Isobutavan	166.67	Mean:        CV:	32	18	6	14	17	32	26	45	141	128
			38	18	16	24	20	32	30	51	146	134
			38	27	18	31	21	43	33	55	147	141
			36	21	13	23	19	36	30	50	145	134
			10	25	48	40	11	18	12	10	2	5
Isobutavan	500.00	Mean:        CV:	24	17	6	13	18	31	18	51	116	136
			29	17	12	30	20	40	30	60	164	140
			36	29	17	31	20	41	36	79	165	140
			30	21	12	25	19	37	28	63	148	139
			20	33	47	41	6	15	33	23	19	2
Isobutavan	1500.0	Mean:        CV:	32	17	7	14	14	30	20	30	125	96
			38	25	15	17	18	36	24	42	126	110
			49	28	21	27	19	39	26	50	140	135
			40	23	14	19	17	35	23	41	130	114
			22	24	49	35	16	13	13	25	6	17
Positive controls		Mean:        CV:	305	213	104	76	241	280	98	200	458	618
			357	315	112	78	353	376	101	283	569	631
			398	326	169	87	***	376	113	313	660	675
			353	285	128	80	297	344	104	265	562	641
			13	22	28	7	27	16	8	22	18	5

S9: Liver homogenate from rats treated with Aroclor

Mean: Average number of revertants per plate when 3 plates are counted

CV: Coefficient of variation (SD/Mean*100)

VC: Viable count

***: Plate not counted due to extravagant results

Conclusions:

It was concluded that Isobutavan did not show any mutagenic activity in the Salmonella/mammalian microsome mutagenicity test under the conditions employed in this evaluation.

Executive summary:

The potential mutagenicity of Isobutavan was investigated in an Ames test performed to GLP and OECD 471. The study is deficient with respect to the current version of the OECD guideline in that a E. coli or S. typhimurium TA102 strain is not included in the study design. S. typhimuriumstrains TA98, TA100, TA1535, TA1537 and TA1538 were exposed in triplicate to Isobutavan at concentrations of up to 1500 µg/plate in the absence and presence of metabolic activation (Aroclor-induced rat hepatic S9 fraction). Isobutavan did not cause an increase in the number of revertant colonies, either in the presence or absence of metabolic activation. Results obtained with the positive control confirmed the validity of the assay. It is therefore concluded that there was no indication of mutagenicity under the conditions of this assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 February 2016 - 05 April 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: In Vitro Mammalian Cell Gene Mutation Test With L5178y Mouse Lymphoma Cells

Specific details on test material used for the study:

- Name of test material (as cited in study report): Isobutavan
- Physical state: Almost colourless to pale yellow liquid
- Lot/batch No.: SC00016272
- Expiration date of the lot/batch: 11 December 2017
- Storage condition of test material: At room temperature protected from light

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

In the first experiment, ISOBUTAVAN was tested up to concentrations of 490 and 1000 μg/ml in the absence and presence of S9-mix, respectively.
In the second experiment, the test item was tested up to concentrations of 450 μg/ml in the absence of S9-mix.

Vehicle / solvent:

The test item was dissolved in dimethyl sulfoxide (DMSO, Merck Darmstadt, Germany).

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Remarks:

with and without S9 activation

Details on test system and experimental conditions:

Dose range finding test:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 17 to 1600 μg/ml in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, an increase above the positive threshold of MF(controls) + 126 (GEF= 237 x 10-6) was observed at the toxic top dose of 490 μg/ml. The increase observed was at a RTG of 10% and according to the guideline a result would not be considered positive if the increase in MF occurred only at or below 10% RTG. The increase observed in the next dose level of 436 μg/ml with a RTG of 32%, was a 1.9-fold increase in the mutation frequency and was not above the GEF.

After the prolonged treatment period (24 hour), an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml with a RTG of 12%. The mutation frequency at this concentration was not above the GEF and there was no concentration related increase observed. Therefore the result is considered to be negative and of no biological relevance.

In the presence of S9-mix, ISOBUTAVAN did not induce a significant increase in the mutation frequency. Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the absence of S9-mix is only observed at the top concentration with a RTG of 10% after the 3 hour treatment and the increase was above the critical value of 170 per 106 survivors after the prolonged treatment period but not above the GEF, the biological relevance of these increases is doubtful. Therefore the test results for the 3hr test in the absence of S9-mix is considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the presence of S9-mix.

Conclusions:

The test item was not mutagenic in the current test in the presence of S9-mix.

Executive summary:

Evaluation of the mutagenic activity of ISOBUTAVAN in an in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. This report describes the effects of ISOBUTAVAN on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hours treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone). The study procedures described in this report were based on the most recent OECD guideline. Batch SC00016272 of ISOBUTAVAN was an almost colourless to pale yellow liquid. The test item was dissolved in dimethyl sulfoxide. In the first experiment, ISOBUTAVAN was tested up to concentrations of 490 and 1000 μg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. The relative total growth (RTG) was 10% in the absence of S9-mix. In the presence of S9-mix, no cytotoxicity was observed, however ISOBUTAVAN was tested up to the precipitating dose level of 1000 μg/ml in compliance with the guideline. In the absence of S9-mix, an increase above the positive threshold of MF(controls) + 126 (GEF= 237 x 10-6) was observed at the toxic top dose of 490 μg/ml with a RTG of 10%. The mutation frequency observed in the second highest dose level of 436 μg/ml (RTG of 32%) was not above the GEF and therefore considered negative. In the presence of S9-mix, none of the tested concentrations reached a mutation frequency of MF(controls) + 126. In the second experiment, the test item was tested up to concentrations of 450 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 12%. After the prolonged treatment period, an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml. The increase observed in the first experiment (short treatment) was at a RTG of 10% and according to the guideline a result would not be considered positive if the increase in MF occurred only at or below 10% RTG. After the prolonged treatment period (24 hour), an increase in the mutation frequency above the critical value of 170 per 106 survivors was only observed at the toxic top dose of 450 μg/ml with a RTG of 12%. The mutation frequency at this concentration was not above the GEF and there was no concentration related increase observed. Therefore the result is considered to be negative and of no biological relevance. The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Since the meaningful increase (above the positive threshold, GEF) in the mutation frequency at the TK locus in the absence of S9-mix is only observed at the top concentration with a RTG of 10% after the 3 hour treatment and the increase was above the critical value of 170 per 106 survivors after the prolonged treatment period but not above the GEF, the biological relevance of this increase is doubtful. Therefore the test results for the 3hr test in the absence of S9-mix are considered equivocal under the experimental conditions described in the report. The test item was not mutagenic in the current test in the presence of S9-mix.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 October 2014 to 30 January 2015

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

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): Isobutavan
- Physical state: Clear almost colourless to pale yellow viscous liquid
- Lot/batch No.: SC00010048
- Expiration date of the lot/batch: December 17, 2015
- Storage condition of test material: In refrigerator (2-8°C) protected from light

Species / strain / cell type:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital and ß-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

In the first cytogenetic assay, Isobutavan was tested up to 950 μg/ml in the absence and up to 975 μg/ml in the presence of S9-fraction for a 3 hours exposure time with a 27 hours harvest time.
In the second cytogenetic assay, Isobutavan was tested up to 500 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix.

Vehicle / solvent:

The vehicle for the test substance was dimethyl sulfoxide.

Untreated negative controls:

yes

Remarks:

Solvent

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine (CAS No. 64-86-8)

Remarks:

with and without metabolic activation

Details on test system and experimental conditions:

Dose range finding test :

In order to select the appropriate dose levels for the in vitro micronucleus test cytotoxicity data was obtained in a dose range finding test. Isobutavan was tested in the absence and presence of S9-mix.
Lymphocytes (0.4 ml blood of a healthy male donor was added to 5 ml or 4.8 ml culture medium, without and with metabolic activation respectively and 0.1 ml (9 mg/ml) Phytohaemagglutinin) were cultured for 46 ± 2 hours and thereafter exposed to selected doses of Isobutavan for 3 hours and 24 hours in the absence of S9-mix or for 3 hours in the presence of S9-mix. Cytochalasine B was added to the cells simultaneously with the test substance at the 24 hours exposure time. A vehicle control was included at each exposure time. The highest tested concentration was determined by the solubility of Isobutavan in the culture medium. After 3 hours exposure to Isobutavan in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasine B (5 μg/ml) and incubated for another 24 hours (1.5 times normal cell cycle). The cells that were exposed for 24 hours in the absence of S9-mix were not rinsed after exposure but were fixed immediately. Cytotoxicity of Isobutavan in the lymphocyte cultures was determined using the cytokinesis-block proliferation index (CBPI index). Based on the results of the dose range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level showed a cytotoxicity of 55 ± 5% whereas the cytotoxicity of the lowest dose level was approximately the same as the cytotoxicity of the solvent control.

First cytogenetic assay:

Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Isobutavan for 3 hours in the absence and presence of S9-mix. After 3 hours exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium with Cytochalasin B (5 μg/ml) and incubated for another 24 hours. Appropriate vehicle and positive controls were included in the first cytogenetic assay.

Second cytogenetic assay:

To confirm the results of the first cytogenetic assay a second cytogenetic assay was performed with an extended exposure time of the cells in the absence of S9-mix.
Lymphocytes were cultured for 46 ± 2 hours and thereafter exposed in duplicate to selected doses of Isobutavan with cytochalasin B (5 μg/ml) for 24 hours in the absence of S9-mix. Appropriate vehicle and positive controls were included in the second cytogenetic assay

Key result

Species / strain:

lymphocytes: human lymphocytes.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Dose range finding test

At a concentration of 1600 μg/ml Isobutavan precipitated in the culture medium. In the dose range finding study, at the 3 hours exposure time, blood cultures were treated with 17, 52, 164, 512 and 1600 μg Isobutavan/ml culture medium with and without S9-mix. At the 24 hours exposure time blood cultures were treated with 17, 52, 164, 512 and 1600 μg Isobutavan/ml culture medium without S9-mix.

First cytogenetic assay

Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay: Without & With S9-mix: 100, 500, 600, 800, 1000, 1200 and 1400 μg/ml culture medium (3 hours exposure time, 27 hours harvest time). No appropriate dose levels could be selected for scoring of micronuclei in absence and presence of S9-mix, since at the concentration of 800 μg/ml not enough cytotoxicity was observed (37 and 34% respectively), whereas the next higher concentration of 1000 μg/ml was too toxic for scoring (76 and 65% respectively). The experiment was repeated in cytogenetic assay 1A. Based on the results of the first cytogenetic assay the following dose levels were selected for the cytogenetic assay: Without & With S9-mix: 100, 800, 825, 850, 875, 900, 925, 950, 975, 1000 and 1050 μg/ml culture medium (3 hours exposure time, 27 hours harvest time). The following dose levels were selected for scoring of micronuclei:Without S9-mix : 100, 825 and 950 μg/ml culture medium (3 hours exposure time, 27 hours harvest time). With S9-mix : 100, 850 and 975 μg/ml culture medium (3 hours exposure time, 27 hours harvest time).

Second cytogenetic assay

To obtain more information about the possible clastogenicity and aneugenicity of Isobutavan, a second cytogenetic assay was performed in which human lymphocytes were exposed for 24 hours in the absence of S9-mix. The following dose levels were selected for the second cytogenetic assay: Without S9-mix : 100, 300, 400, 450, 500, 525, 550 and 600 μg Isobutavan/ml culture medium (24 hours exposure time, 24 hours harvest time).

The following dose levels were selected for the scoring of micronuclei: Without S9-mix : 100, 400 and 500 μg Isobutavan/ml culture medium (24 hours exposure time, 24 hours harvest time).

Conclusions:

Finally, it is concluded that this test is valid and that Isobutavan is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

An in vitro micronucleus assay with Isobutavan in cultured peripheral human lymphocytes. This report describes the effect of Isobutavan on the number of micronuclei formed in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix). The possible clastogenicity and aneugenicity of Isobutavan was tested in two independent experiments. The study procedures described in this report were based on the most recent OECD and EC guidelines. Batch SC00010048 of Isobutavan was a clear almost colourless to pale yellow viscous liquid with a purity of 98.7%. The test substance was dissolved in dimethyl sulfoxide. In the first cytogenetic assay, Isobutavan was tested up to 950 μg/ml in the absence and up to 975 μg/ml in the presence of S9-fraction for a 3 hours exposure time with a 27 hours harvest time. Isobutavan precipitated in the culture medium at these dose levels. Appropriate toxicity was reached at these dose levels. In the second cytogenetic assay, Isobutavan was tested up to 500 μg/ml for a 24 hours exposure time with a 24 hours harvest time in the absence of S9-mix. In addition appropriate toxicity was reached at this dose level. 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, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. Isobutavan did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei in the absence and presence of S9-mix, in either of the two experiments. Finally, it is concluded that this test is valid and that Isobutavan is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in this report.