dimethyl 1,2-benzenedicarboxylate;reaction mass of: 1,2-dimethylpropylidene dihydroperoxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of Ames and MLA in vitro studies it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay or in the the mouse lymphoma L5178Y test system. The cells were exposed to MIKP(in DMP) in DMSO. An in vitro assay for chromosome aberration is not available for the registered substance. Data from a structural analogue is used to for this endpoint, for further information see the attached read across justification in section 13. They key study for MEKP is a mammalian cell cytogenetic assay for chromosome aberration. V79 cell cultures were exposed to MEKP (in TXIB/diacetone alcohol) in DMSO. There was no evidence of chromosome aberration induced over background.

There are also in vitro studies available for MEKP in DMP. These have not been included in this dossier since they were disregarded (assigned a klimisch 3) in the MEKP dossier. In short summary: In the Ames studies MEKP in DMP was negative. In an MLA, sister chromatid exchanges and in vitro chromosome aberration postitive results were seen but only at the highest test concentration where also pronounced cytotoxicity was observed.

The additive DMP is not a mutagenic/clastogenic substance in vivo and has not to classified and labelled with regard to this hazard.

Based on these results further in vivo investigations are not required.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 January 2002 - 31 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine + typtophan operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see below

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: see below

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

1st experiment:
all strains: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix.
2nd experiment:
TA1535, TA1537, TA98 and TA100
With and without S9-mix: 10, 33, 100, 333 and 1000 µg/plate
WP2uvrA
Without S9-mix: 10, 33, 100, 333, 1000 and 2000 µg/plate.
With S9-mix : 10, 33, 100, 333 and 1000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: NA
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan

NUMBER OF REPLICATIONS: At least five different doses (increasing with approximately half-log steps) of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independentc c.q. tryptophan independent) were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present.

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn

Evaluation criteria:

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

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

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

Statistics:

None

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see results table

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see results table

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see results table

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see results table

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS: Not examined

RANGE-FINDING/SCREENING STUDIES: Combined with first experiment

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

In the second experiment in tester strain TA1537, MIPKP induced an up to 2.5-fold increase in the absence of S9-mix. However, this increase was only observed in one experiment and the highest number of revertants was not higher than 20 and within our historical control data range. Therefore, this increase is considered to be not biologically relevant and the test substance is considered to be not mutagenic. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose related, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix).

In the combined range finding test/first mutation assay, MIKP was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. MIPK did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains. In the second mutation assay, it was tested up to concentrations of 1000 µg/plate in the absence and presence of S9-mix in the strains TA1535, TA1537, TA98 and TA100. TRIGONOX R-938 was tested up to concentrations of 2000 and 1000 µg/plate in the absence and presence of S9-mix, respectively in strain WP2uvrA. Toxicity was observed in all tester strains. The presence of 5 and 10% (v/v) liver microsomal activation did not influence these findings.

In the second experiment in tester strain TA1537, MIKP induced an up to 2.5-fold increase in the absence of S9-mix. However, this increase was only observed in one experiment and the highest number of revertants was not higher than 20 and within our historical control data range. Therefore, this increase is considered to be not biologically relevant and the test substance is considered to be not mutagenic.

All other bacterial strains showed negative responses over the entire dose range, i.e. no doserelated, two-fold, increase in the number of revertants in two independently repeated experiments.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that MIPKP is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 March 2002 - 22 April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK)

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:

Experiment 1:
Without S9-mix: 0.5, 1,5, 10,20,30,40,50,60,70,80 and 90 µg/ml exposure medium.
With 8% (v/v) S9-mix: 1, 10, 30, 50, 70, 100, 130, 175, 230, 275 and 300 µg/ml exposure medium.

Experiment 2:
Without 59-mix: 10,50,75,100,130,175,200,225,250,275,300 and 325 µg/ml exposure medium.
With 12% (v/v) 59-mix: 10, 50, 100, 130, 175, 225, 250, 275, 300, 325 and 350 µg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:no data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Experiment 1: 3 hours
Experiment 2: 24 hours
- Expression time (cells in growth medium):
Experiment 1: 3 days
Experiment 2: 2 days
- Selection time (if incubation with a selection agent): 10-11 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14-15 days

SELECTION AGENT (mutation assays): TK

NUMBER OF REPLICATIONS: Single cultures per test concentration with independent repeat.

NUMBER OF CELLS EVALUATED:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological dense colonies with a hazy contour and with a diameter larger than a quarter of a well.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls was ≥ 50%.
b) In at least seven of the eight doses of the test substance, an acceptable number of surviving cells (10E6) could be analysed for expression of the TK mutation.
c) The spontaneous mutant frequency in the untreated or solvent control was < 10 per 10E5 clonable cells.
d) The positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant (at least three-fold) increases in the mutant frequencies.

Statistics:

The experimental results were not subjected to statistical analysis.
A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and
b) The results were reproducible in an independently repeated test.

A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

see table

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see table

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test substance concentration range of 1 to 333 µg/ml in the absence of S9-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.
In the absence of S9-mix after 3 hours of treatment, no toxicity in the suspension growth was observed up to concentrations of 33 µg/ml compared to the suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 and 333 µg/ml after 3 hours treatment.
In the presence of S9-mix after 3 hours of treatment, no toxicity in the suspension growth was observed up to concentrations of 100 µg/ml compared to the suspension growth of the solvent control. No cell survival was observed at the test substance concentration of 333 µg/ml after 3 hours treatment and 24 hours of subculture.
In the absence of S9-mix after 24 hours of treatment, no toxicity in the suspension growth was observed up to concentrations of 100 µg/ml compared to the suspension growth of the solvent control. No cell survival was observed at the test substance concentration of 333 µg/ml after 24 hours of treatment.

COMPARISON WITH HISTORICAL CONTROL DATA: Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: None

The spontaneous mutant frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range ({0.7- and 6.3 x 10E5 (mean 2.7 x 10E5 ) in the absence of S9-mix} and {0.7- and 6.9 x 10E5 (mean 3.1 x 10E5 ) in the presence of S9mix}; for n=164 and 151 respectively).

Mutant frequencies in cultures treated with positive control chemicals were increased by 11- and 40-fold for EMS in the first and second experiment respectively, and by 13- and 10-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system

(S9-mix) functioned properly.

Mutant frequency at the TK-Iocus

The test substance did not induce a significant increase in the mutant frequency in the absence or presence of S9 metabolic activation in the first experiment. This result was confirmed in a second, repeat experiment with modifications in the duration of treatment in the absence of S9, and in the composition of the S9 concentration for metabolic activation.

Conclusions:

Interpretation of results (migrated information):
negative

In conclusion, MIPKP is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

This report describes the effects of MIPKP on the induction of forward mutations at the thymidine-kinase locus (TK-Iocus) in L5178Y mouse lymphoma cells in the presence and absence of S9-mix. The test was performed in two independent experiments in the presence and absence of S9-mix (Aroclor-1254 induced rat liver S9-mix). A range finding study was performed to set dose levels for the subsequent mutation studies and to establish the solubility of MIPKP. The test substance precipitated in the exposure medium at a test substance concentration of 325 µg/ml. In the first experiment, it was tested up to concentrations of 90 and 300 µg/ml in the absence and presence of 8 % (v/v) S9-mix, respectively. Incubation time was 3 hours.

Appropriate toxicity was observed at these dose levels in the absence and presence of S9-mix. In the second experiment, MIPKP was tested up to concentrations of 225 and 350 µg/ml in the absence and presence of 12 % (v/v) S9-mix, respectively. Incubation times were 24 hours and 3 hours for incubations in the absence and presence of S9 metabolic activation respectively. Toxicity was observed at the dose level of 225 µg/ml in the absence of S9-mix. In the presence of S9-mix, it was tested beyond the limit of the solubility and showed no toxicity at this dose level.

Mutant frequencies in cultures treated with positive control chemicals were increased by 11- and 40-fold for EMS in the first and second experiment respectively, and by 13- and 10-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly.

MIPKP did not induce a significant increase in the mutant frequency in the absence or presence of S9 metabolic activation in the first experiment. This result was confirmed in a second, repeat experiment with modifications in the duration of treatment in the absence of S9, and in the composition of the S9 concentration for metabolic activation.

It is concluded that MIPKP is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See attached justification in section 13

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: DME
- Properly maintained: yes
- Checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

mammalian microsomal fraction (S9 mix)

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 9.76, 19.53, 29.29, 39.06, 58.59 µg/mL
with S9 mix: 9.76, 19.53, 29.29, 39.06, 58.59 µg/mL

Experiment B with 20/20 h treatment/sampling time
without S9 mix: 4.88, 9.76, 19.53, 39.06, 58.59 µg/mL
Experiment B with 20/28 h treatment/sampling time
without S9 mix: 4.88, 9.76, 19.53, 39.06, 58.59 µg/mL
Experiment B with 3/28 h treatment/sampling time
with S9 mix: 4.88, 9.76, 19.53, 39.06, 58.59, 78.12 µg/mL

Vehicle / solvent:

- Solvent used: DMSO
- Justification for choice of solvent: no data; however, DMSO is a standard solvent used in studies of this type and a concurrent solvent control was included.

Untreated negative controls:

yes

Remarks:

Dulbecco's Modified Eagle's (DME) medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO in DME medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with meatbolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 3 or 20 hours
- Expression time (cells in growth medium): 20 or 28 hours
- Fixation time (start of exposure up to fixation or harvest of cells): after 20 or 28 hours (after expression time)

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa stain (5 %)

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED: at least 200 metaphase cells containing 2 N ± 2 centromeres

DETERMINATION OF CYTOTOXICITY
- Method: % cells in relation to solvent control

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data.
– the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.

Statistics:

A Chi square test was performed.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: no precipitation was noted for examined test concentrations

RANGE-FINDING/SCREENING STUDIES:
Based on the results of a pre-test (cytotoxicity) the concentrations indicated in section "Test concentrations" were selected

COMPARISON WITH HISTORICAL CONTROL DATA:
The observed chromosome aberration rates were within the ranges of historical control data.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results (migrated information):
negative

The test item was non-clastogenic in this system.

Executive summary:

In a mammalian cell cytogenetic assay for chromosome aberration, V79 cell cultures were exposed to methyl-ethylketone peroxide (in TXIB and diacetone alcohol) in DMSO at (i) 9.76, 19.53, 29.29, 39.06 and 58.59 µg/mL (without and without S9 mix) with 3 hours treatment and 20 hours sampling time, at (ii) 4.88, 9.76, 19.53, 39.06 and 58.59 µg/mL (without S9 mix) with 20 hours treatment and 20 hours sampling time or with 20 hours treatment and 28 hours sampling time and at (iii) 4.88, 9.76, 19.53, 39.06, 58.59 and 78.12 µg/mL with 3 hours treatment and 28 hours sampling time. The results of a pretest on cytotoxicity were used as basis for dose level selection. Methyl-ethylketone peroxide was tested up to cytotoxic concentrations with and without metabolic activation. Positive controls induced the appropriate responses. There was no evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for test guideline OECD 473 for in vitro cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance substance is not mutagenic in the Ames test or the MLA. A structural analogue is negative in an in vitro chromosome aberration assay. Therefore the test substance is not classified for genetic toxicity.