Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1. In vitro gene mutation study in bacteria（AMES）: Negative（OECD 471）

2. In vitro cytogenicity study in mammalian cells：Negative（OECD 473）

3. In vitro gene mutation study in mammalian cells(HPRT Assay)：Negative (OECD 476)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Remarks:

Only TA97,TA98,TA100 and TA102 tested during the study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Version:1998

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Species / strain / cell type:

S. typhimurium TA 102

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 97

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared according to the method of Ams in department of genetic toxicology of IOM,CAMP and stored at -80℃ for use.

Test concentrations with justification for top dose:

According to results of inhibition test on bacteria TA 100, the dosages of test substance were 1000 μg/plate,500 μg/plate,250 μg/plate,125 μg/plate and 62.5 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile deionized water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

methylmethanesulfonate

other: 2,4,7-trinitro-9-flourenone and 2-hydroxy anthraquinone

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:triplicate
- Number of independent experiments:1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10*8
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:16 hours
- Exposure duration/duration of treatment:48 hours

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

other: not available as the study was conducted according to old version of Test Guideline

Remarks:

not available

Cytotoxicity / choice of top concentrations:

other: not available as the study was conducted according to old version of Test Guideline

Vehicle controls validity:

other: not available as the study was conducted according to old version of Test Guideline

Untreated negative controls validity:

other: not available as the study was conducted according to old version of Test Guideline

True negative controls validity:

other: not available as the study was conducted according to old version of Test Guideline

Positive controls validity:

other: not available as the study was conducted according to old version of Test Guideline

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No clear signs of cytotoxicity and precipitation were observed.Compared with the historical data, the numbers of reverent colonies per plate of blank and positive control for the 4 bacterial strains were all in the acceptable ranges.Neither a concentration-related increase over the range tested and a reproducible increase at one or more concentrations in the number of reverent colonies per plate in at least on strain with and withour metabolic activation system were found.

Conclusions:

The substanse is not mutagenic in the Salmonella Typhimurium.

Executive summary:

In a Bacterial Reverse Mutation Test according to OECD TG 471(version:1998), TA 97, TA98 TA100 and TA 102 were utilized to detect the gene mutation potential of the test item in the absence and presence of S9 activation system.No clear signs of cytotoxicity and precipitation were observed.Compared with the historical data, the numbers of reverent colonies per plate of blank and positive control for the 4 bacterial strains were all in the acceptable ranges.Neither a concentration-related increase over the range tested and a reproducible increase at one or more concentrations in the number of reverent colonies per plate in at least on strain with and withour metabolic activation system were found. The substanse is considered as not mutagenic in the Salmonella Typhimurium.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)

Version / remarks:

1998

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
S9 was prepared according to the method of Ames in department of genetic toxicology of IOM,CAPM and store at -80 celsius for use.

Test concentrations with justification for top dose:

In cell growth inhibition test, the 50% cell growth inhibition concentration was 360 μg/mL which was used as the highest dose for aberration test and other 4 treatment doses were180 μg/mL,90 μg/mL,45 μg/mL and 22 μg/mL.

Vehicle / solvent:

sterile deionized water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration:duplicate
- Number of independent experiments:1

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:24 hours
- Exposure duration/duration of treatment: 24 hours and 48 hours

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

No clear signs of cytotoxicity and precipitation were observed.The frequencies of cells with structural chromosome aberration in 5 dose levels of test substance were all less than 3%.Neither a statistically significant and reproducible positive response at any test points nor a statistically significant dose-related increase in the number of chromosome aberrations were found.

Conclusions:

The substance does not induce chromosome aberrations in cultured Chinese H mster Lung fibroblast cells.

Executive summary:

In a Chromosomsl aberration study in Chinese Hamster Lung Fibroblast cell study according to OECD TG 473(version:1998), cells were exposed to test item with a dose level of 360 μg/mL,180 μg/mL,90 μg/mL,45 μg/mL and 22 μg/mL  to detect the chromosome abbrration potential of the test item in the absence and presence of S9 activation system. No clear signs of cytotoxicity and precipitation were observed.The frequencies of cells with structural chromosome aberration in 5 dose levels of test substance were all less than 3%.Neither a statistically significant and reproducible positive response at any test points nor a statistically significant dose-related increase in the number of chromosome aberrations were found.The substance does not induce chromosome aberrations in cultured Chinese H mster Lung fibroblast cells.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Name: Reaction Product of 2-Hydroxybenzoic Acid, Styrene and Oxozinc
Batch No. 2011121802
EC No. 700-321-2 (provisional EC number)
Aggregate State at room temperature:Solid
Color:Amber color
Odor: Slight odor
Molecular Formula: C30H26O6Zn, C46H42O6Zn, C62H58O6Zn,
Molecular Weight: 547.93 to 964.53 g/mol
Purity: 100 % (UVCB-Substance)
Date of Certificate of Analysis: June 04, 2013
Analysis (Method): HPLC/MS
Date of Manufacture: December 18, 2011
Stability: Stable under storage conditions as indicated
Storage conditions:0 – 50℃
Expiry Date: December 04, 2013
Safety precautions:Routine safety precautions will be applied (lab coat, mask, gloves, safety glasses)
Identification: The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item, Certificate of Analysis, MSDS and Test Item Characterisation Sheet were supplied by the Sponsor and archived with the raw data.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CHO KI: Sub-line (KI) of Chinese hamster ovary cell line CHO
Lot. No.: 09J019
Supplier: ECACC (European Collection of Cell Cultures)
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO KI is a sub-line of CHO cell line. The CHO KI cell line was purchased from ECACC (European Collection of Cell Cultures).

The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file. For each experiment more vials were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. When cells were growing well, subcultures were established in an appropriate number of flasks. The CHO KI cells for this study were grown in Ham's F12 medium (F12-10) supplemented with 1 % (v/v) of Antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 % (v/v)). During the 5 and 20 hour treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (v/v) (F12-5). The selection medium for TG resistant mutants contained 20 μM of 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

Metabolic activation:

with and without

Metabolic activation system:

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Kerkrader Strasse 10, D-35394 Giessen, Germany; manufacturer: MOLTOX INC., P.O. BOX 1189, BOONE, NC 28607 USA).

Test concentrations with justification for top dose:

Experiment 1, 5-hour treatment period without S9 mix:
30, 40, 50, 60, 65 and 70 and μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 95, 1001 and 1101 μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
27.5, 30, 32.5, 35, 37.5 and 40 μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 95, 1001 and 1101 μg/mL

Treatment concentrations for the mutation assay were selected on the basis of the result of a Pre-test on cell toxicity. A dose selection (cytotoxicity assay) was performed. During the cytotoxicity assay, 1-3 day old cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells were seeded into 90 mm petri dishes (tissue culture quality: TC sterile) at 106 cells each and incubated in culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in Ham's F12-10 medium in absence or in presence (11 concentrations) of S9 mix (50 μL/mL) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. Additional groups of cells were treated for 20 hours without metabolic activation (16 concentrations). 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-0 medium, covered with trypsin-EDTA solution and then counted. The cell concentration was adjusted to 40 cells/mL with Ham's F12-10 medium. For each dose, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air for 5-7 days for colony growing. Colonies were then fixed with methanol, stained with Giemsa and the colonies were counted. Survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was examined visually at beginning and end of the treatments.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Details on test system and experimental conditions:

A 5-hour treatment in the presence and absence of S9 mix was performed. For the 5-hour treatment, 106 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each concentration, for the negative (solvent) controls and the positive controls for treatment without and with S9 mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. The exposure period was 5 hours. Following the exposure period the cells were washed with F12-0 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-0 medium and detached with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
In samples where sufficient cells survived, cell number was adjusted to 105 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

Five hours treatment in the presence of S9 mix and 20-hour treatment in the absence of S9 mix were performed. Duplicate cultures were used for each treatment. For the treatment, 106 cells were placed in each of a series of sterile dishes (diameter approx. 10 mm) and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. 24 hours after the beginning of treatment the cells were washed with F12-0 medium and were detached with trypsin-EDTA solution, counted and after this, the cells were subcultured to assess cytotoxicity and to begin the phenotypic expression period.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
• Mutant frequency between the negative (solvent) and the test item or positive control item treated groups.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Comparable toxicity was observed in treatment groups when compared to the negative (solvent) controls, both in the absence and in the presence of the metabolic activation, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 c

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. In the test item treated groups.

In Experiment 2, the mutant frequencies of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures. The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory.
The mutation frequency (MF) of the positive control item (EMS) after the 5-hours incubation was slightly above the corresponding historical control data range, however within the assay acceptance range. The slightly higher mutation frequency was considered as acceptable and did not have any effect on the validity of the test.
The pH and osmolality of control and treatment solutions did not show any alterations compared to the concurrent control groups in Experiments 1 and Experiment 2 .

Conclusions:

It is concluded that the test item, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc, was not mutagenic in this in vitro mammalian cell gene mutation test performed with in Chinese hamster ovary cells.

Executive summary:

The test item, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in Dimethyl sulfoxide (DMSO) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix).
Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:
Experiment 1, 5-hour treatment period without S9 mix:
30, 40, 50, 60, 65 and 70 and μg/mL
Experiment 1, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 95, 1001 and 1101 μg/mL
Experiment 2, 20-hour treatment period without S9 mix:
27.5, 30, 32.5, 35, 37.5 and 40 μg/mL
Experiment 2, 5-hour treatment period with S9 mix:
50, 60, 70, 80, 95, 1001 and 1101 μg/mL
1:These concentrations were very toxic and there were not enough cells to start the phenotypic expression period after the treatment.
Phenotypic expression was evaluated up to 8 days following exposure.
In Experiment 1, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no biologically significant differences between treatment and control groups and no dose-response relationships were noted.
In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency.
As in Experiment 1, no statistical differences between treatment and solvent control groups and no dose-response relationships were noted in Experiment 2. The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures.
Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was not mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

1. In vitro gene mutation study in bacteria（AMES）: Negative（OECD 471）

In a Bacterial Reverse Mutation Test according to OECD TG 471(version:1998), TA 97, TA98 TA100 and TA 102 were utilized to detect the gene mutation potential of the test item in the absence and presence of S9 activation system.No clear signs of cytotoxicity and precipitation were observed.Compared with the historical data, the numbers of reverent colonies per plate of blank and positive control for the 4 bacterial strains were all in the acceptable ranges.Neither a concentration-related increase over the range tested and a reproducible increase at one or more concentrations in the number of reverent colonies per plate in at least on strain with and withour metabolic activation system were found. The substanse is considered as not mutagenic in the Salmonella Typhimurium. 

 

2. In vitro cytogenicity study in mammalian cells：Negative（OECD 473）

In a Chromosomsl aberration study in Chinese Hamster Lung Fibroblast cell study according to OECD TG 473(version:1998), cells were exposed to test item with a dose level of 360 μg/mL,180 μg/mL,90 μg/mL,45 μg/mL and 22 μg/mL  to detect the chromosome abbrration potential of the test item in the absence and presence of S9 activation system. No clear signs of cytotoxicity and precipitation were observed.The frequencies of cells with structural chromosome aberration in 5 dose levels of test substance were all less than 3%.Neither a statistically significant and reproducible positive response at any test points nor a statistically significant dose-related increase in the number of chromosome aberrations were found.The substance does not induce chromosome aberrations in cultured Chinese H mster Lung fibroblast cells.

 

3. In vitro gene mutation study in mammalian cells(HPRT Assay)：Negative (OECD 476)

The test item, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in Dimethyl sulfoxide (DMSO) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9 mix). Two independent main experiments (both run in duplicate) were performed.The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large increases in mutation frequency in the positive control cultures. Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was not mutagenic under the conditions of this study.

Justification for classification or non-classification

Based on the available information in the dossier, the substance Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc does not need to be classified for germ cell mutagenicity when the criteria outlined in CLP Regulation (Annex I of 1272/2008/EC) are applied.