4-methylstyrene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 479 (Genetic Toxicology: In Vitro Sister Chromatid Exchange Assay in Mammalian Cells)

Deviations:

no

GLP compliance:

no

Type of assay:

sister chromatid exchange assay in mammalian cells

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

without

Test concentrations with justification for top dose:

Trial 1: 1, 10, 25, 50 and 100 nL/mL
Trial 2: 25, 50, 65, 80 and 100 nL/mL (confirmatory assay)
Trial 3: 25, 50, 65, 80 and 100 nL/mL (repeating Trial 2 with new lot of test substance due to suspected improper storage of lot used in both earlier
assays)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, 1%

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

0.1 µgmL

Details on test system and experimental conditions:

Whole blood cultures
Human venous blood was drawn into a sterile syringe that contained heparin to prevent clotting. Cultures were initiated by adding 0.6 ml of blood to 9.4 ml pf medium in 25 cm2 plastic flasks standing upright to minimize the surface area. The medium was RPMI 1640 supplemented with 15 % fetal calf serum (FCS), 1% penicillin-streptomycin, and phytohaemagglutinin (PHA-M).
Treatment of cultures with the test substance
Blood was added to culture medium containing PHA and incubated at 37 °C in the dark for 24 h. At this time cells are beginning to grow due to stimulation by PHA. Solutions of the test substance EMS or the solvent control were then added to appreciate cultures, followed by BrdU (final concentration 25 uM) and cultures were re-incubated for a further 46-48 hours (total culture time: 70-72 h). Colcemid was added 2.5 hours before harvest of dividing lymphocytes. Two replicate cultures were used for each treatment in assay.
Lymphocyte fixation
Thee cell suspension was centrifuged, the supernatant discarded, and cells treated with hypotonic KCl (0.075 M) for 3 minutes to swell the cells and get rid of red blood cells. Cells were then washed three times with fixative (methanol/glacial acetic acid, (3:1)) and dropped on to slides to air day. In trial 3 BrdU was added at initiation of cultures, not after the test compound.
Staining for detection of SCE was accomplished by a modified fluorescent plus Giemsa technique (Perry and Wolff 1974 and Goto et al. 1978).

Key result

Species / strain:

lymphocytes: Human

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The solvent control did not show any mutagenic activity. Results for the positive control showed expected mutagenic activity.
Trial 1: A slight, but statistically significant (P<0.001) higher number of sister chromatid exchanges (SCEs) per cell was noted at 50 and 100 nL/mL compared to solvent control, with some evidence of a dose-response.
Trial 2: This assay repeated the first trial, using more concentrations in the range of ‘positives’ from Trial 1. In this assay a slight, but statistically significant (P<0.25 or P<0.001) higher number of SCEs per cell was observed at 50 through 80 nL/mL. However, the difference from solvent control at the highest concentration of 100 nL/mL was not statistically significant and, overall, there was no evidence of a dose-response.
Trial 3: A new lot of the test substance, stored at -20⁰C (instead of room temperature), was used in this trial, using the same concentrations of test substance as in Trial 2 and following essentially the same methodology. A slight, but statistically significant (P<0.001) higher number of SCEs per cell was noted in this assay at concentrations of 65 through 100 nL/mL, without a clear dose-relationship.   
Dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay.

Treatment	Mitotic index (% 500 cells)	% of cells at first second and third metaphase
Solvent control	1.2 %	26	58	16
25 nl/ml	0.4 %	42	51	7
100 nl/ml	0.6 %	66	34	0

Conclusions:

Under the study conditions, dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay. A slight but statistically significant and reproducible higher number of SCEs was observed in cultured human lymphocytes treated with test substance over a concentration range of 50 to 100 nL/mL. The maximum response in cultures treated with test material was approximately 84% over the solvent control. Given that these data did not meet the evaluation criteria for a ‘positive’ result, with a less than a 2-fold increase in SCEs over solvent control and without a clear dose-response, the test substance was designated as ‘weakly positive’.

Executive summary:

A study using cultured human lymphocytes was conducted to determine the potential of the test substance to induce sister chromatid exchanges (SCEs) in vitro, according to a method similar to OECD Guideline 479. DMSO (1%) was used as the solvent control and the positive control was ethylmethanesulfonate. Three trials were performed, using an overall concentration range of 1 to 100 nL/mL. The solvent control did not show any activity, results for the positive control showed expected increases in SCEs. Under the study conditions, dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay. A slight but statistically significant and reproducible higher number of SCEs was observed in cultured human lymphocytes treated with test substance over a concentration range of 50 to 100 nL/mL. The maximum response in cultures treated with test material was approximately 84% over the solvent control. Given that these data did not meet the evaluation criteria for a ‘positive’ result, with a less than a 2-fold increase in SCEs over solvent control and without a clear dose-response, the test substance was designated as ‘weakly positive’ (Galloway, 1980).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

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:

no

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Additionally TA1538 and Saccharomyces cerevisiae D4

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Sprague-Dawley rat liver

Test concentrations with justification for top dose:

0.001, 0.01, 0.1, 1.0 and 5.0 µL/plate, with and without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Details on test system and experimental conditions:

Indicator organisms:
Salmonella typhimurium: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Saccharomyces cerevisiae: D4

S-9 homogenate: from Aroclor 1254-treated male Sprague-Sawley rats (5 days’ treatment)

Positive controls:
Non-activation: Methylnitrosoguanidine, 2-Nitrofluorene, Quinacrine mustard
Activation: 2-Anthramine, 2-Acetylaminofluorine, 8-Aminoquinoline

Plate Test (overlay method):
Approximately 10+E08 cells from an overnight culture of each indicator strain were added to separate test tubes containing 2.0 ml of molten agar supplemented with biotin and a trace of histidine. For activation and non-activation tests, at least four dose levels were tested. Just prior to purring, an aliquot of reaction mixture (0.5 ml containing the 9.000 x g liver homogenate) was added to each of the activation overlay tubes, which were then mixed, and the contents poured over the surface of a mineral agar plate and allowed to solidify. The plates were incubated for 48 h at 37 °C, and scored for the number of colonies growing on each plate. Positive controls were included for assays run with and without metabolic activation.

The test substance was evaluated using a concentration range of 0.001, 0.01, 0.1, 1.0 and 5.0 microlitres per plate (toxicity was noted at 5.0 microlitres per plate for strain TA 1537).

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

One strain at 5 µl (TA 1537)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The assays performed both in the presence and absence of the metabolic activation system were negative (no increase in revertant colonies).

Conclusions:

Under the study conditions, the assays performed both in the presence and absence of the metabolic activation system were negative (no increase in revertant colonies). The test substance did not demonstrate mutagenic activity in any of the assays conducted in this study, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic under the test conditions.

Executive summary:

A study was conducted to determine the in vitro mutagenicity of the test substance according to a method similar to OECD Guideline 471 (Ames assay). The substance was evaluated using the tester strainsSalmonella typhimuriumTA 1535, TA 1537, TA 1538, TA 98 and TA 100, and Salmonella cerevisiae, D4. S9 homogenate from Aroclor 1254-treated male Sprague-Dawley rats (5 day treatment) was used to provide a metabolic activation system. Positive controls were incorporated into the study design. For non-activation assays, methylnitrosoguanidine, 2-nitrofluorene and quinacrine mustard were used. For activation assays, 2-anthramine, 2-acetylaminofluorine and 8-aminoquinoline were used. The test substance was evaluated at a concentration range of 0.001, 0.01, 0.1, 1.0 and 5.0 µl/plate (toxicity was noted at 5.0 µl/plate for strain TA 1537). Under the study conditions, the assays performed both in the presence and absence of the metabolic activation system were negative (no increase in revertant colonies). The test substance did not demonstrate mutagenic activity in any of the assays conducted in this study, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic under the test conditions (Jagannath, 1977).

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 481 (Genetic Toxicology: Saccharomyces cerevisiae, Mitotic Recombination Assay)

Deviations:

no

GLP compliance:

no

Type of assay:

mitotic recombination assay with Saccharomyces cerevisiae

Species / strain / cell type:

Saccharomyces cerevisiae

Additional strain / cell type characteristics:

other: Strain D5

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced Sprague-Dawley rat liver

Test concentrations with justification for top dose:

0.01, 0.1, 0.5, 1.0, 2.0 and 2.5 µL/plate, with and without metabolic activation

Vehicle / solvent:

DMSO (1%) was used as the solvent control.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

Indicator cells: Saccharomyces cerevisiae, strain D5
S-9 homogenate: from Aroclor 1254-treated male Sprague-Dawley rats
Assays were performed with and without a metabolic activation system, using concentrations of 0.01, 0.1, 0.5, 1.0, 2.0 and 2.5 µL test material / plate.

Key result

Species / strain:

Saccharomyces cerevisiae

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

The solvent control did not show any mutagenic activity. Results for the positive controls showed expected mutagenic activity.
The assays performed on the test substance, both in the presence and absence of the metabolic activation system, were positive in this evaluation.
The test substance exhibited activity with strain D5 of Saccharomyces cerevisiae and was shown to induce mitotic recombinations with and without metabolic activation.
The total events per 10+E05 survivors exceeded the spontaneous conversion rate by greater than two fold at 0.5, 1.0, 2.0 and 2.5 µL/plate with and without metabolic activation

Conclusions:

Under the study conditions, the assays performed on the test substance both in the presence and absence of the metabolic activation system were positive. The test substance exhibited activity with strain D5 of Saccharomyces cerevisiae and was shown to induce mitotic recombinations. The total events per 10E+5 survivors exceeded the spontaneous conversion rate by greater than two fold at 0.5, 1.0, 2.0 and 2.5 µL/plate with and without metabolic activation.

Executive summary:

A study was conducted to determine the mutagenicity of the test substance in the mitotic recombination assay using Saccharomyces cerevisiae, strain D5, both in the absence and presence of a metabolic activation system, according to method similar to OECD Guideline 481. Concentrations of 0.01, 0.1, 0.5, 1.0, 2.0 and 2.5 µL/plate were used, with and without metabolic activation. The solvent control (DMSO at 1%) did not show mutagenic activity. Results for the positive controls showed expected mutagenic activity. Under the study conditions, the assays performed on the test substance both in the presence and absence of the metabolic activation system were positive. The test substance exhibited activity with strain D5 of Saccharomyces cerevisiae and was shown to induce mitotic recombinations. The total events per 10E+5 survivors exceeded the spontaneous conversion rate by greater than two fold at 0.5, 1.0, 2.0 and 2.5 µL/plate with and without metabolic activation (Jagannath, 1980).

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Not specified

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Male mouse liver

Test concentrations with justification for top dose:

0.02, 0.03, 0.04, 0.06 and 0.08 µL/mL, with and without metabolic activation

Vehicle / solvent:

DMSO (1%) was used as the solvent control and growth medium without addition of solvent was included as a negative control.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

dimethylnitrosamine (2nd +ive control)

Details on test system and experimental conditions:

Indicator cells: Fischer mouse lymphoma cell line derived from L5178Y. The cells are heterozygous for a specific autosomal mutation at the TK locus are bromodeoxyuridine sensitive. Socoring for mutation was based on selecting cells that have undergone forward mutation from a TK +/- to a TK-/- genotype by cloning them in soft agar with BUdR.

The cells were maintained in Fishers medium for Leukemic cells of Mice with 10 % horse serum and sodium pyruvate. Cloning medium consisted of Fisher’s medium with 20% horse serum, sodium pyruvate, and 0.37% agar. Selection medium was made from cloning medium by the addition of 5.0 mg of BUdR to 100 ml of cloning medium.

The experimental design included determination of the solubility and toxicity of the test substance prior to selecting concentrations for the assays.
Assays were performed with and without a metabolic activation system, using concentrations of 0.02, 0.03, 0.04, 0.06 and 0.08 mcg test substance per mL.
Positive controls: non-activation: Ethylmethanesulfonate, activation: Dimethylnitrosamine.

Non-activation assay and activation assay: the procedure used is a modification of that reported by Clive and Spector (Mutation Research 31:17-29, 1975).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

0.02, 0.03, 0.04, 0.06 and 0.08 µg

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

0.12 µg/mL and higher

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Screening assays showed that the test substance was completely cytotoxic to cells at concentrations of 0.12 µg/mL and higher.
The solvent control and negative control did not show any mutagenic activity. Results for the positive controls showed expected mutagenic activity.
The assays performed on the test substance, both in the presence and absence of the metabolic activation system, were negative; there was no evidence of mutagenicity either in the absolute number of mutants or in the mutation frequency observed. 

Conclusions:

Under the study conditions, the test substance, did not demonstrate any mutagenic activity, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic in the mouse lymphoma assay.

Executive summary:

A study was conducted to determine potential of the test substance to induce specific locus forward mutations in the L5178Y Thymidine Kinase mouse lymphoma cell assay, according to a method comparable to OECD Guideline 476. A Fischer mouse lymphoma cell line derived from L5178Y was used. DMSO served as solvent control and growth medium without addition of solvent was included as negative control. The positive control for the non-activation assay was ethylmethanesulfonate, with dimethylnitrosamine as the positive control for the activation assay. Assays were performed with and without a metabolic activation system, using concentrations of 0.02, 0.03, 0.04, 0.06 and 0.08 µg test substance/ml. Screening assays showed that the substance was completely cytotoxic to cells at concentrations of 0.12 µg/mL and higher. The solvent control and negative control did not show any mutagenic activity. Results for the positive controls showed expected mutagenic activity. Under the study conditions, the test substance, did not demonstrate any mutagenic activity, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic in the mouse lymphoma assay (Matheson, 1977).

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

disregarded due to major methodological deficiencies

Study period:

From May 01, 1980 to October 02, 1980

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: Pre-OECD 477

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 477 (Genetic Toxicology: Sex-linked Recessive Lethal Test in Drosophila melanogaster)

Deviations:

yes

GLP compliance:

yes

Type of assay:

Drosophila SLRL assay

Species:

Drosophila melanogaster

Sex:

male/female

Route of administration:

inhalation: aerosol

Dose / conc.:

0.07 other: ml

Key result

Sex:

not specified

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Under the study conditions, treatment with the test substance did not increase the frequencies of exceptions over those of controls in any of the assays. It appears that the test substance does not induce significant genetic damage in Drosophila melanogaster.

Executive summary:

A study was conducted to determine the potential mutagenicity of the test substance in the Drosophila mutagenicity assay. Exposure of flies to 0.07 ml carried as an aerosol into a 25 ml flask with immediate removal resulted in highly aberrant patterns of activity approaching anesthetization. The treatment resulted in about 20% mortality with some 30% of the survivors being sterile. Longer exposures resulted in almost total mortality. Under the study conditions, treatment with the test substance did not increase the frequencies of exceptions over those of controls in any of the assays. It appears that the test substance does not induce significant genetic damage in Drosophila melanogaster (Bowman, 1980).

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

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 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Breeding Farms, Wilmington, USA
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Not specified (125-175 g on arrival at 4-6 weeks)
- Housing: 5 per cage
- Diet (e.g. ad libitum): Purina rat chow, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 11 days

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Olive oil
- Concentration of test substance in vehicle: Dose volume for all groups 4 mL/kg

Details on exposure:

Three groups of 5 males were given the test substance, by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg/day in olive oil; total dose volume, 4 mL/kg. A further group of 5 males was given olive oil (negative control) by oral gavage at 4.0 mL/kg/day for 5 consecutive days. A fifth group of 5 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg by intraperitoneal injection (IP), one day prior to sacrifice.
 
All rats were given colchicine at 4 mg/kg IP 4 hours prior to sacrifice. The rats were sacrificed by CO2 asphyxiation, the femurs removed and the marrow flushed into Hanks’ Balanced Salt Solution (HBSS). After centrifugation at 800-1000 rpm for 8-10 minutes, the cells were treated with 0.075 M KCl for 20-30 minutes at 37 ± 2 ⁰C. The cells were then re-centrifuged and washed twice with 5 mL Carnoy’s fixative. The cells were resuspended in 5 mL Carnoy’s fixative and treated overnight (16-20 hrs) at 4 ± 2 ⁰C. The cells were centrifuged at 800-1000 rpm for 8-10 minutes and the cell pellet was resuspended to opalescence in fresh Carnoy’s fixative. Two to five slides were prepared for each animal and stained with Giemsa and permanently mounted.
 
A minimum of 50 metaphase spreads from each animal was examined and scored for chromatid and chromosomal gaps and breaks, fragmentation, structural rearrangements, and ploidy. The mitotic index was recorded for each animal as the number of cells in mitosis/100 cells observed.

Duration of treatment / exposure:

5 days

Frequency of treatment:

Once daily for 5 days (positive control, one dose only)

Post exposure period:

Animals sacrificed the day after last dose

Dose / conc.:

0.15 other: mL/kg bw/day

Remarks:

nominal

Dose / conc.:

0.5 other: mL/kg bw/day

Remarks:

nominal

Dose / conc.:

1.5 other: mL/kg bw/day

Remarks:

nominal

No. of animals per sex per dose:

5 groups of 5 males (Control, 3 doses of test substance, positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine
- Justification for choice of positive control(s): Not specified, but included in OECD 474
- Route of administration: Intraperitoneal injection, once only
- Doses / concentrations: 0.5 mg/kg (vehicle/concentration not given)

Evaluation criteria:

The percentage of cells in the negative control group showing chromatid and chromosome breaks and gaps must be ≤ 3% of the total cells
evaluated. The percentage of cells showing aberrations of any type must not exceed 10% in the negative control group. In the positive control group, the number of cells with aberrations must be at least 45% of the total cells evaluated.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There were no adverse effects or mortalities attributable to treatment with the test substance.
There was no difference in ploidy between groups given the test substance and that given the negative control. The test substance was not a mitotic inhibitor when compared to negative control; the positive control was shown to be a moderate mitotic inhibitor at the dose administered.
The percentage of cells with aberrations in groups given the test substance was not statistically significantly (P>0.05) increased relative to negative control. The positive control group showed severe effects, with approximately 54% of all cells having one or more aberrations.
The total aberrations per cell in groups given the test substance was not statistically significantly (P>0.05) increased relative to negative control. The positive control group showed severe effects, with approximately 2.11 aberrations per cell.

Conclusions:

Under the study conditions, treatment of males with the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg/day did not result in any evidence of clastogenic potential in this assay.

Executive summary:

A study was conducted to determine mutagenic potential of the test substance according to a method similar to OECD Guideline 474. Three groups of 5 males were given the test substance, by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day in olive oil, total dose volume, 4 mL/kg bw. A further group of 5 males was given olive oil (negative control) by oral gavage at 4.0 mL/kg bw/day for 5 consecutive days. A fifth group of 5 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg bw by intraperitoneal injection (IP), one day prior to sacrifice. All rats were given colchicine at 4 mg/kg bw i.p. 4 h prior to sacrifice. The rats were sacrificed, the femurs removed and the marrow obtained. After manipulation, two to five slides were prepared for each animal and stained with Giemsa and permanently mounted. A minimum of 50 metaphase spreads from each animal was examined and scored for chromatid and chromosomal gaps and breaks, fragmentation, structural rearrangements, and ploidy. The mitotic index was recorded for each animal as the number of cells in mitosis/100 cells observed. There were no adverse effects or mortalities attributable to treatment with the test material. There was no difference in ploidy, the percentage of cells with aberrations or the total aberrations per cell between groups given the test material and that given the negative control. The positive control was shown to be a moderate mitotic inhibitor at the dose administered; approximately 54% of all cells had one or more aberrations, with approximately 2.11 aberrations per cell. Under the study conditions, treatment of males with the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day did not result in any evidence of clastogenic potential in this assay (Putman, 1979).

Reference 3

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)

GLP compliance:

no

Type of assay:

rodent dominant lethal assay

Specific details on test material used for the study:

Lot/batch No.: 03248001-B1 & 03248001-B2

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Breeding Farms, Quebec, Canada
- Age at study initiation: 10-12 weeks
- Weight at study initiation: Males, 265-370 g; Females, not specified
- Assigned to test groups: Randomly; basis not specified
- Housing: Males, 3 per cage during acclimation; one per cage thereafter. Females, 5 per cage during acclimation; 2-4 per cage after mating. Plastic cages with hardwood chip bedding
- Diet: Certified laboratory rodent chow, ad libitum
- Water: ad libitum
- Acclimation period: 10-14 days
- Temperature: Approximately 23 °C
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle: Olive oil, for controls only

Details on exposure:

Three groups of 10 males were given the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg/day. A further group of 10 males was given olive oil (negative control) by oral gavage at 1.0 mL/kg/day for 5 consecutive days. A fifth group of 10 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg by intraperitoneal injection. Three days after the last dose, each male was mated with 2 virgin females over a 5-day period. Males were then allowed to rest for 2 days. Subsequently, the mating process was repeated with additional virgin females until the males had been mated for 7 weeks, 2 females per week.
14-15 days from the mid-point of the mating period, the females were sacrificed by CO2 asphyxiation. The abdominal cavity was exposed and the membrane removed from each ovary. The corpora lutea were counted for each ovary and recorded separately. The uterine contents were examined and the number of live and dead implants was recorded for each uterine horn. The criteria for a living foetus were viability at necropsy, general colour, and absence of maceration; a dead implant was that present at the implantation site as a non-viable foetus reduced in size or as a necrotic embryonic mass. Males were sacrificed after completion of the mating period, but no necropsies were performed.

Duration of treatment / exposure:

Males only, 5 days

Frequency of treatment:

Males, once daily for 5 days (positive control, one dose only)

Post exposure period:

Males mated for 7 weeks post-exposure

Dose / conc.:

0.15 other: mL/kg bw/day

Dose / conc.:

0.5 other: mL/kg bw/day

Dose / conc.:

1.5 other: mL/kg bw/day

No. of animals per sex per dose:

5 groups of 10 males (Control, 3 doses of test substance, positive control), plus females for mating.

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine
- Justification for choice of positive control(s): Not specified, but included in OECD 478
- Route of administration: Intraperitoneal injection, once only
- Doses / concentrations: 0.5 mg/kg bw in distilled water (concentration not given)

Evaluation criteria:

Criteria for determination of a valid test:
Female Sprague-Dawley rats mated with negative control males must show an average of no less than 8 implantations per pregnant female. Females mated to positive control males must exhibit severe foetal damage, i.e. a statistically significant reduction in implantations relative to the negative controls and a statistically significant increase in females with 2 or more dead implants relative to the negative controls. This damage must be noted between weeks 2 and 7 of the spermatogenic cycle.

Statistics:

Dominant lethality was evaluated according to the following criteria, which were analysed statistically by recognised methods:
Fertility Index (the number of fertile matings)
Total implantations per pregnant female
Number of corpora lutea per pregnant female
Pre-implantation losses per pregnant female (difference between the number of corpora lutea and the number of total (live and dead) implantations
Dead implants per pregnant female
The proportion of pregnant females with one or more dead implants, and with two or more dead implants
Dead implants per total implants
Live implants per pregnant female

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Other than body weight losses / reduced weight gain in males given the test substance, particularly at 0.5 and 1.5 mL/kg/day, there were no remarkable clinical findings.

Additional information on results:

Other than body weight losses / reduced weight gain in males given the test substance, particularly at 0.5 and 1.5 mL/kg/day, there were no remarkable clinical findings. The pregnancy data for females mated with males previously dosed with the positive control, triethylenemelamine, revealed expected findings, including: markedly reduced implantations for matings performed in weeks 1-4, and, for matings performed in weeks 1-5, markedly increased pre-implantation losses, increased number of dead implants per pregnancy, increased proportion of pregnant females with one/two or more dead implants, increased number of dead implants per total implants, and reduced number of live implants per pregnant female. Comparison of the pregnancy data for females mated with males previously dosed with the test substance with that of females mated with males given the negative control did not reveal any differences attributable to treatment with the test substance.

Conclusions:

Under the study conditions, treatment of males with the test substance and their subsequent mating over a 7 week period (covering the spermatogenic cycle) with untreated virgin females did not result in any evidence of mutagenic potential.

Executive summary:

A study was conducted to determine the cytogenicity and chromosome aberration potential of the test substance according to a method similar to OECD Guideline 478. Three groups of 10 male Sprague Dawley rats were given the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day. A further group of 10 males was given olive oil (negative control) by oral gavage at 1.0 mL/kg bw/day for 5 consecutive days. A fifth group of 10 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg bw by intraperitoneal injection. Three days after the last dose, each male was mated with 2 virgin females over a 5 d period. Males were then allowed to rest for 2 d. Subsequently, the mating process was repeated with additional virgin females until the males had been mated for 7 weeks, 2 females per week. Fourteen to fifteen days from the mid-point of the mating period, the females were sacrificed by CO2 asphyxiation. The abdominal cavity was exposed and the membrane removed from each ovary. The corpora lutea were counted for each ovary and recorded separately. The uterine contents were examined and the number of live and dead implants was recorded for each uterine horn. The criteria for a living foetus were viability at necropsy, general colour, and absence of maceration; a dead implant was that present at the implantation site as a non-viable foetus reduced in size or as a necrotic embryonic mass. Males were sacrificed after completion of the mating period, but no necropsies were performed. The pregnancy data for females mated with males previously dosed with the positive control revealed expected findings. Comparison of the pregnancy data for females mated with males previously dosed with the test substance with that of females mated with males given the negative control did not reveal any differences attributable to treatment with the test substance. Under the study conditions, treatment of males with the test substance and their subsequent mating over a 7 week period (covering the spermatogenic cycle) with untreated virgin females did not result in any evidence of mutagenic potential (Putman, 1980).

Reference 4

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From October 20, 1980 to December 02, 1980

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 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

yes

Remarks:

Study was performed according to a protocol similar to OECD Guideline 475

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

- Source: Taconic Farms, Germantown, USA
- Weight at study initiation: Not specified (188 +/- 27 g on arrival)
- Assigned to test groups: Randomly, based on a table of random numbers
- Housing: 1 per cage
- Diet (e.g. ad libitum): Purina Rodent Chow 5001, ad libitum
- Water (e.g. ad libitum): ad libitum
- Temperature (°C): Approximately 22.2 °C
- Humidity (%): 30-75
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.75% Aqueous Methocel K4M Premium (hydroxypropyl methylcellulose)
- Concentration of test substance in vehicle: Dose volume for all groups 10 mL/kg
- Amount of vehicle (if gavage or dermal): As above

Duration of treatment / exposure:

5 d

Frequency of treatment:

Once daily for 5 d (positive control, one dose only)

Post exposure period:

Animals sacrificed the day after last dose

Dose / conc.:

0.134 other: g/kg bw/day

Remarks:

nominal

Dose / conc.:

0.45 other: g/kg bw/day

Remarks:

nominal

Dose / conc.:

1.34 other: g/kg bw/day

Remarks:

nominal

No. of animals per sex per dose:

5 groups of 5 males (Control, 3 doses of test substance, positive control)

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): Not specified, but included in OECD 475
- Route of administration: Oral, by gavage, once only
- Doses / concentrations: 60.0 mg/kg in vehicle (6.0 mg/mL)

Evaluation criteria:

Criteria for determination of a valid test:
Chi squared analysis was performed to compare test values to those for negative controls. A statistically significant increase above control in aberrant cells was considered indicative of clastogenic activity by the test substance.

Statistics:

Recognised methods.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not examined

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Slight oral discharge and decreased activity were noted for some animals at the highest dose of the test substance. Slight nasal discharge was noted for one animal at the mid dose, with decreased activity in one animal at the low dose. There were no other notable clinical findings.
 
Analysis of the blood sample obtained 2 hours after the last dose revealed the following approximate concentration of the test substance:
 
1.34 g/kg/day: 26.5 mcg/mL
0.45 g/kg/day: 14.0 mcg/mL
0.134 g/kg/day: 1.5 mcg/mL
Negative control: 0.0 mcg/mL
 
An increase in the number of aberrant cells was not observed in animals given the test substance; there was no evidence of clastogenicity under the conditions of this study. The positive control yielded the expected high rate of aberrant cells. 

Conclusions:

Under the study conditions, an increase in the number of aberrant cells was not observed in animals given the test substance and no evidence of clastogenicity noted. The positive control yielded the expected high rate of aberrant cells.

Executive summary:

A study was conducted to determine whether the test substance was capable of causing a significant increase in clastogenic events in the bone marrow of rats in vivo, according to a protocol similar to OECD Guideline 475. The study comprised 5 groups of 5 male Sprague-Dawley rats for chromosome analysis. Three groups were given the test substance by oral gavage for 5 consecutive days at doses of 1.34, 0.45 or 0.134 g/kg bw/day in the vehicle, Methocel K4M Premium (0.75% aqueous hydroxypropyl methylcellulose) total dose volume, 10 mL/kg bw. A further group of 5 males was given the vehicle only (negative control) by oral gavage at 10 mL/kg bw/day for 5 consecutive days. A fifth group of 5 males was given a single dose of the positive control (cyclophosphamide) at 60.0 mg/kg bw by oral gavage in the vehicle (10 mL/kg bw), 1 d prior to sacrifice. For all groups, colchicine was given IP (4 mg/kg bw) 4 h after the last dose and 2 h prior to sacrifice the following day. At sacrifice, one femur was taken from each animal and processed for metaphase analysis. A minimum of 5 slides were prepared for each animal; 50 cells were examined for each animal for the presence of clastogenic effects. Under the study conditions, an increase in the number of aberrant cells was not observed in animals given the test substance and no evidence of clastogenicity noted. The positive control yielded the expected high rate of aberrant cells (Schreiner, 1980).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

A study was conducted to determine the in vitro mutagenicity of the test substance according to a method similar to OECD Guideline 471 (Ames assay). The substance was evaluated using the tester strains Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, and Salmonella cerevisiae, D4. S9 homogenate from Aroclor 1254-treated male Sprague-Dawley rats (5 day treatment) was used to provide a metabolic activation system. Positive controls were incorporated into the study design. For non-activation assays, methylnitrosoguanidine, 2-nitrofluorene and quinacrine mustard were used. For activation assays, 2-anthramine, 2-acetylaminofluorine and 8-aminoquinoline were used. The test substance was evaluated at a concentration range of 0.001, 0.01, 0.1, 1.0 and 5.0 µl/plate (toxicity was noted at 5.0 µl/plate for strain TA 1537). Under the study conditions, the assays performed both in the presence and absence of the metabolic activation system were negative (no increase in revertant colonies). The test substance did not demonstrate mutagenic activity in any of the assays conducted in this study, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic under the test conditions (Jagannath, 1977).

A study was conducted to determine the mutagenicity of the test substance in the mitotic recombination assay using Saccharomyces cerevisiae, strain D5, both in the absence and presence of a metabolic activation system, according to method similar to OECD Guideline 481. Concentrations of 0.01, 0.1, 0.5, 1.0, 2.0 and 2.5 µL/plate were used, with and without metabolic activation. The solvent control (DMSO at 1%) did not show mutagenic activity. Results for the positive controls showed expected mutagenic activity. Under the study conditions, the assays performed on the test substance both in the presence and absence of the metabolic activation system were positive. The test substance exhibited activity with strain D5 of Saccharomyces cerevisiae and was shown to induce mitotic recombinations. The total events per 10E+5 survivors exceeded the spontaneous conversion rate by greater than two fold at 0.5, 1.0, 2.0 and 2.5 uL/plate with and without metabolic activation (Jagannath, 1980).

A study was conducted to determine potential of the test substance to induce specific locus forward mutations in the L5178Y Thymidine Kinase mouse lymphoma cell assay, according to a method comparable to OECD Guideline 476. A Fischer mouse lymphoma cell line derived from L5178Y was used. DMSO served as solvent control and growth medium without addition of solvent was included as negative control. The positive control for the non-activation assay was ethylmethanesulfonate, with dimethylnitrosamine as the positive control for the activation assay. Assays were performed with and without a metabolic activation system, using concentrations of 0.02, 0.03, 0.04, 0.06 and 0.08 µg test substance/ml. Screening assays showed that the substance was completely cytotoxic to cells at concentrations of 0.12 µg/mL and higher. The solvent control and negative control did not show any mutagenic activity. Results for the positive controls showed expected mutagenic activity. Under the study conditions, the test substance, did not demonstrate any mutagenic activity, either in the presence or absence of the metabolic activation system, and was therefore considered not to be mutagenic in the mouse lymphoma assay (Matheson, 1977).

A study using cultured human lymphocytes was conducted to determine the potential of the test substance to induce sister chromatid exchanges (SCEs) in vitro, according to a method similar to OECD Guideline 479. DMSO (1%) was used as the solvent control and the positive control was ethylmethanesulfonate. Three trials were performed, using an overall concentration range of 1 to 100 nL/mL. The solvent control did not show any activity, results for the positive control showed expected increases in SCEs. Under the study conditions, dose-related cell toxicity was noted at all test substance concentrations, as evidenced by a reduction in the frequency of dividing cells (mitotic index) and cell cycle delay. A slight but statistically significant and reproducible higher number of SCEs was observed in cultured human lymphocytes treated with test substance over a concentration range of 50 to 100 nL/mL. The maximum response in cultures treated with test material was approximately 84% over the solvent control. Given that these data did not meet the evaluation criteria for a ‘positive’ result, with a less than a 2-fold increase in SCEs over solvent control and without a clear dose-response, the test substance was designated as ‘weakly positive’ (Galloway, 1980).

In vivo

A study was conducted to determine the cytogenicity and chromosome aberration potential of the test substance according to a method similar to OECD Guideline 478. Three groups of 10 male Sprague Dawley rats were given the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day. A further group of 10 males was given olive oil (negative control) by oral gavage at 1.0 mL/kg bw/day for 5 consecutive days. A fifth group of 10 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg bw by intraperitoneal injection. Three days after the last dose, each male was mated with 2 virgin females over a 5 d period. Males were then allowed to rest for 2 d. Subsequently, the mating process was repeated with additional virgin females until the males had been mated for 7 weeks, 2 females per week. Fourteen to fifteen days from the mid-point of the mating period, the females were sacrificed by CO2 asphyxiation. The abdominal cavity was exposed and the membrane removed from each ovary. The corpora lutea were counted for each ovary and recorded separately. The uterine contents were examined and the number of live and dead implants was recorded for each uterine horn. The criteria for a living foetus were viability at necropsy, general colour, and absence of maceration; a dead implant was that present at the implantation site as a non-viable foetus reduced in size or as a necrotic embryonic mass. Males were sacrificed after completion of the mating period, but no necropsies were performed. The pregnancy data for females mated with males previously dosed with the positive control revealed expected findings. Comparison of the pregnancy data for females mated with males previously dosed with the test substance with that of females mated with males given the negative control did not reveal any differences attributable to treatment with the test substance. Under the study conditions, treatment of males with the test substance and their subsequent mating over a 7 week period (covering the spermatogenic cycle) with untreated virgin females did not result in any evidence of mutagenic potential (Putman, 1980).

A study was conducted to determine whether the test substance was capable of causing a significant increase in clastogenic events in the bone marrow of rats in vivo, according to a protocol similar to OECD Guideline 475. The study comprised 5 groups of 5 male Sprague-Dawley rats for chromosome analysis. Three groups were given the test substance by oral gavage for 5 consecutive days at doses of 1.34, 0.45 or 0.134 g/kg bw/day in the vehicle, Methocel K4M Premium (0.75% aqueous hydroxypropyl methylcellulose) total dose volume, 10 mL/kg bw. A further group of 5 males was given the vehicle only (negative control) by oral gavage at 10 mL/kg bw/day for 5 consecutive days. A fifth group of 5 males was given a single dose of the positive control (cyclophosphamide) at 60.0 mg/kg bw by oral gavage in the vehicle (10 mL/kg bw), 1 d prior to sacrifice. For all groups, colchicine was given IP (4 mg/kg bw) 4 h after the last dose and 2 h prior to sacrifice the following day. At sacrifice, one femur was taken from each animal and processed for metaphase analysis. A minimum of 5 slides were prepared for each animal; 50 cells were examined for each animal for the presence of clastogenic effects. Under the study conditions, an increase in the number of aberrant cells was not observed in animals given the test substance and no evidence of clastogenicity noted. The positive control yielded the expected high rate of aberrant cells (Schreiner, 1980).

A study was conducted to determine mutagenic potential of the test substance according to a method similar to OECD Guideline 474. Three groups of 5 males were given the test substance, by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day in olive oil, total dose volume, 4 mL/kg bw. A further group of 5 males was given olive oil (negative control) by oral gavage at 4.0 mL/kg bw/day for 5 consecutive days. A fifth group of 5 males was given a single dose of the positive control (triethylenemelamine) at 0.5 mg/kg bw by intraperitoneal injection (IP), one day prior to sacrifice. All rats were given colchicine at 4 mg/kg bw i.p. 4 h prior to sacrifice. The rats were sacrificed, the femurs removed and the marrow obtained. After manipulation, two to five slides were prepared for each animal and stained with Giemsa and permanently mounted. A minimum of 50 metaphase spreads from each animal was examined and scored for chromatid and chromosomal gaps and breaks, fragmentation, structural rearrangements, and ploidy. The mitotic index was recorded for each animal as the number of cells in mitosis/100 cells observed. There were no adverse effects or mortalities attributable to treatment with the test material. There was no difference in ploidy, the percentage of cells with aberrations or the total aberrations per cell between groups given the test material and that given the negative control. The positive control was shown to be a moderate mitotic inhibitor at the dose administered; approximately 54% of all cells had one or more aberrations, with approximately 2.11 aberrations per cell. Under the study conditions, treatment of males with the test substance by oral gavage for 5 consecutive days at doses of 0.15, 0.5 or 1.5 mL/kg bw/day did not result in any evidence of clastogenic potential in this assay (Putman, 1979).

Justification for classification or non-classification

The substance was tested in a number of in vitro assays. The outcome was negative in all but one, a non-standard in vitro mitotic recombination assay using Saccharomyces cerevisiae. An ambiguous/weakly positive response was also obtained in a sister chromatid exchange assay. The substance was then further evaluated in several in vivo assays, all of which yielded negative outcomes. Overall, the weight of evidence suggests that the test substance is not genotoxic and therefore does not warrant classification for this endpoint according to CLP (EC 1272/2008) criteria.