Dimethyl heptanedioate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): mutagenic in presence of metabolic activation with the pre-incubation method in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA102.

- MLA test (OECD 492, GLP, K, rel 1): Non audited results clearly tends to negative results on L5178Y TK+/- mouse lymphoima cells - On going study

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:

16 November 2017 - 11 January 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD test guideline No. 471 and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted on 21st July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

French GLP Compliance Programme for chemical products (inspected on 13-15 April 2016 / signed on 02 November 2016)

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium: Histidine gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

S. typhimurium TA 100

Remarks:

Third experiment

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

No

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix (10% v/v)

Test concentrations with justification for top dose:

Preliminary test: with and without S9-mix: 10, 100, 500, 1000, 2500 and 5000 µg/plate ; direct plate incorporation method (tested in the TA 98, TA 100 and TA 102 strains, one plate/dose level)
First test: with and without S9-mix: 312.5, 625, 1250, 2500 and 5000 µg/plate ; direct plate incorporation method
Second test: without S9-mix: 312.5, 625, 1250, 2500 and 5000 µg/plate ; direct plate incorporation method / with S9-mix: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate ; pre-incubation method
Third test: with S9-mix: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate ; direct plate incorporation method and pre-incubation method

The highest dose level will be selected according to the following criteria specified in international regulations and on the basis of the data obtained in the preliminary toxicity test:
- for non-toxic, freely soluble test items, the highest dose level will be 5000 μg/plate,
- for non-toxic, poorly soluble test items, the highest dose level will be at least the lowest precipitating dose level,
- for toxic test items, irrespective of solubility, the highest dose level will be based on the level of toxicity: clearing of the bacterial lawn and/or reduction in the number of revertants when compared to the vehicle controls. However, precipitation should not interfere with the scoring of the test.

Since the test item was found freely soluble in the final treatment medium and non-toxic in the preliminary test, the highest dose level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Not specified

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9 mix, 1 µg/plate in DMSO for strains TA100 and TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without S9 mix, 50 µg/plate in DMSO for strain TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9 mix, 0.5 µg/plate in DMSO for strain TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

Without S9 mix, 0.5 µg/plate in water for strain TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

With S9 mix, 2 µg/plate in DMSO for strains TA1535, TA1537 and TA98

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

With S9 mix, 20 µg/plate in DMSO for strain TA102

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9 mix, 5 µg/plate in DMSO for strain TA100

Details on test system and experimental conditions:

TEST SYSTEM: The five strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 were supplied by Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) or Culture Collections (Public Health England, Porton Down, Salisbury, SP4 0JG, UK). These bacterial strains are stored in cryoprotective medium (1 mL nutrient broth and 0.09 mL dimethylsulfoxide) at -80°C. The day before treatment, cultures were inoculated from frozen permanents. Cultures were seeded under sterile conditions into approximately 13 mL of nutrient broth. The nutrient broth was then placed under agitation in an incubator at 37°C for about 12 hours, to produce bacterial suspensions.

METHOD OF APPLICATION: three independent tests
- First test: direct plate incorporation method (with and without S9-mix)
- Second test: direct plate incorporation method (without S9-mix) and pre-incubation method (with S9-mix)
- Third test: direct plate incorporation method and pre-incubation method (with S9-mix for both tests)

DURATION :
- Preincubation period: 60 minutes at 37°C with shaking
- Exposure duration: 48 to 72 hours of incubation at 37°C

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The evaluation of the toxicity was performed on the basis of the observation of a decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

ACCEPTANCE CRITERIA:
Each main experiment was considered valid if the following criteria are fully met:
- the mean number of revertants in the vehicle controls is consistent with our historical data, in each strain and test condition,
- at least five analyzable dose levels (i.e. including at least three non-cytotoxic dose levels) are obtained for each strain and test condition,
- the mean number of revertants in the positive controls is higher than that of the vehicle controls (at least 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or at least 3-fold increase (for the TA 1535 and TA 1537 strains)).

Evaluation criteria:

In all cases, biological relevance (such as reproducibility and reference to historical data) was taken into consideration when evaluating the results.

The test item is considered to have shown mutagenic activity in this study if:
- a reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the mean number of revertants compared with the vehicle controls is observed, in any strain, at any dose level,
- and/or a reproducible dose-response relationship is evidenced.

The test item is considered to have shown no mutagenic activity in this study if:
- neither an increase in the mean number of revertants, reaching 2-fold (for the TA 98, TA 100 and TA 102 strains) or 3-fold (for the TA 1535 and TA 1537 strains) the vehicle controls value, is observed at any of the tested dose levels,
- nor any evidence of a dose-response relationship is noted.

Statistics:

None

Key result

Species / strain:

S. typhimurium, other: TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitate was observed in the Petri plates when scoring the revertants at any dose levels, either with or without S9 mix.

RANGE-FINDING/SCREENING STUDIES:
Using a test item concentration of 100 mg/mL in the vehicle (i.e. DMSO) and a treatment volume of 50 µL/plate, the highest recommended dose level of 5000 µg/plate was achievable. Thus, the dose levels selected for the preliminary test were: 10, 100, 500, 1000, 2500 and 5000 µg/plate.
In the absence of S9 mix, no noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at any dose levels, in any strains.
In the presence of S9 mix, a moderate thinning of the bacterial lawn was noted at 2500 µg/plate in the TA 98 strain and at 1000 µg/plate in the TA 102 strain. This observation being not dose-related, it was considered not to be biologically relevant and therefore was not taken into consideration for the selection of the highest dose level to be tested in the mutagenicity experiments.

HISTORICAL CONTROL DATA
- The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. The mean revertant colony counts for the vehicle controls were within or close to the current historical control range for the laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY
- Experiments without S9 mix: A moderate toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted at 5000 µg/plate in the TA 98 strain in the second experiment, and at dose levels >= 1666.7 µg/plate in the TA 102 strain in both experiments. No other noteworthy toxicity was noted at any dose levels, in any other strains.
- Experiments with S9 mix: A moderate to strong toxicity (thinning of the bacterial lawn) was noted at dose levels >= 1666.7 µg/plate in the TA 98 strain using both the direct plate incorporation and pre-incubation methods and in the TA 1535 and TA 100 strains using the pre-incubation method only. No other noteworthy toxicity was noted at any dose levels, in any other strains or incubation methods.

MUTAGENICITY RESULTS
In the first experiment using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels, in any of the five strains with or without metabolic activation.

In the second experiment using the pre-incubation method, noteworthy increases in the number of revertants were observed in the TA 98 and TA 100 strains. These increases exceeded the positive threshold of 2-fold the vehicle control value at 5000 µg/plate in the TA 98 strain (3.1-fold the vehicle control value) and at dose levels ≥ 625 µg/plate in the TA 100 strain (up to 6.6-fold the vehicle control value) with an evidence of a dose response relationship. Moreover, the corresponding mean numbers of revertants were above the vehicle control historical range.

A third confirmatory experiment was performed both according to the direct plate incorporation method and pre-incubation method for comparative purposes in the TA 100 strain only.
Using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels in the TA 100 strain.
Using the pre-incubation method, noteworthy increases in the number of revertants were again observed in the TA 100 strain. These increases exceeded the positive threshold of 2-fold the vehicle control value at dose levels ≥ 625 µg/plate (up to 4.8-fold the vehicle control value) with an evidence of a dose response relationship. Moreover, the corresponding mean numbers of revertants were above the vehicle control historical range.

Since the results obtained with the TA 100 strain in the first and second experiments with S9 mix were reproduced in this third experiment, they were considered to be biologically relevant, and to show a clear positive response using the pre-incubation method. As a consequence, checking for reproducibility of the increase observed in the TA 98 strain in the second experiment with S9 mix was considered unneedful and no additional experiment was undertaken.

Remarks on result:

other: Evidence of mutagenic activity

Remarks:

Only with pre-incubation method

None

Conclusions:

Under the experimental conditions of this study, the test item Dimethyl Pimelate showed a mutagenic activity in the presence of a rat liver metabolizing system using the pre-incubation method in the bacterial reverse mutation test with Salmonella typhimurium strains.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and the EU Method B.13/14 and in compliance with GLP, several Salmonella typhimurium strains were treated with the test item using the direct plate incorporation method or the pre-incubation method.

A preliminary toxicity test was performed to define the dose levels of Dimethyl Pimelate, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in a total of three independent experiments. The two first experiments were performed with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. A third experiment was then performed with S9 mix.

The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method (i.e.60 minutes). The third experiment with S9 mix was performed both according to the direct plate incorporation method and pre-incubation method for comparative purposes, at the request of the Sponsor. 

In the two first experiments, five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102, whereas only the TA 100 strain was used in the third experiment (with S9 mix). Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found freely soluble in the final treatment medium and non-toxic in the preliminary test, the highest dose level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analyzable dose levels for each strain and test condition. The study was therefore considered to be valid. No precipitate was observed in the Petri plates when scoring the revertants at any dose levels, either with or without S9 mix.

 

Experiments without S9 mix:

The selected dose levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments. No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants at any dose levels, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

 

Experiments with S9 mix:

A moderate toxicity (thinning of the bacterial lawn) was noted at 5000 µg/plate in the TA 1535, TA 98 and TA 102 strains using the pre-incubation method (i.e.in the second experiment). No other noteworthy toxicity was noted in any other strains or test conditions.

- In the first experiment using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels, in any of the five strains.

- In the second experimentusing the pre-incubation method,noteworthy increases in the number of revertants were observed in the TA 98 and TA 100 strains. These increases exceeded the positive threshold of 2-fold the vehicle control value at 5000 µg/plate in the TA 98 strain (3.1-fold the vehicle control value) and at dose levels ≥ 625 µg/plate in the TA 100 strain (up to 6.6-fold the vehicle control value) with an evidence of a dose-response relationship. Moreover,the corresponding mean numbers of revertants were abovethe vehicle control historical range.

- A third confirmatory experiment was performed both according to the direct plate incorporation method and pre-incubation method for comparative purposes in the TA 100 strain only. 

Using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels in the TA 100 strain.

Using the pre-incubation method, noteworthy increases in the number of revertants were again observed in the TA 100 strain. These increases exceeded the positive threshold of 2-fold the vehicle control value at dose levels ≥ 625 µg/plate (up to 4.8-fold the vehicle control value) with an evidence of a dose‑response relationship. Moreover, the corresponding mean numbers of revertantswere abovethe vehicle control historical range.

 

Since the results obtained with the TA 100 strain in the first and second experiments with S9 mix were reproduced in this third experiment,they were considered to be biologically relevant, and to show a clear positive response using the pre-incubation method. As a consequence,checking for reproducibility of the increase observed in the TA 98 strain in the second experiment with S9 mix was considered unneedful andno additional experiment was undertaken.

Under the experimental conditions of this study, the test item Dimethyl Pimelate showed a mutagenic activity in the presence of a rat liver metabolizing system using the pre-incubation method inthe bacterial reverse mutation test with Salmonella typhimurium strains.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

On going

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline 490 without deviation

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase (tk) gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- cells were obtained from ATCC (American Type Culture Collection, Manassas, USA), through Biovalley (77601, Marne-La-Vallée, France).

The cells are stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at approximately -80°C or in liquid nitrogen.
Each batch of frozen cells is purged of spontaneous TK-/- mutants and checked for the absence of mycoplasma. The cells are maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% (v/v), in a 37°C, 5% CO2 humidified incubator.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction will be purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by the intraperitoneal route. Preserved in sterile tubes at around -80°C until use.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Test item will be dissolved or suspended in the vehicle to provide a suitably concentrated stock solution or suspension. The stock solution/suspension and any further dilutions will be prepared within 4 hours before use, and then kept at room temperature until use

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With S9

Details on test system and experimental conditions:

CELL CUTLRURE: L5178Y TK+/- cells are an established cell line recommended by international regulations for the in vitro mammalian cell gene mutation test. These cells have demonstrated sensitivity to chemical mutagens, a high cloning efficiency and a stable spontaneous mutant frequency. The modal chromosome number is 40, the average cell cycle time is 10-12 hours and the TK phenotypic expression time is 2 days. The cells are stored in a cryoprotective medium [10% horse serum and 10% dimethylsulfoxide (DMSO)] at approximately -80°C or in liquid nitrogen. Each batch of frozen cells is purged of spontaneous TK-/- mutants and checked for the absence of mycoplasma. The cells are maintained in flasks as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 10% (v/v), in a 37°C, 5% CO2 humidified incubator

Rationale for test conditions:

A maximum concentration of 2000 μg/mL was selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to the maximum recommended concentration according to current regulatory guidelines. Concentrations for the Main Experiments were selected based on the results of this cytotoxicity Range-Finder Experiment.

Evaluation criteria:

Evaluation of a positive response:
Based on IWGT recommendations (e, f, g), a test item is considered clearly positive if, in any of the experimental conditions examined:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the global evaluation factor (GEF) of 126 x 10 6,
- a dose-response relationship is demonstrated by a statistically significant trend test.

Evaluation of a negative response:
A test item is considered clearly negative if, in all experimental conditions, no dose-response relationship is demonstrated or, if there is an increase in MF, it does not exceed the GEF.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, will not be considered as positive results.


A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (h):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.

If inconclusive results are observed, any additional confirmatory experiments (at additional cost) will be detailed in a study plan amendment.

Statistics:

To assess the dose-response relationship, a linear regression will be performed between dose-levels and individual mutation frequencies obtained from cultures showing a mean Adj. RTG ≥ 10%. This statistical analysis will be performed using SAS Enterprise Guide software.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

other: on going

Cytotoxicity / choice of top concentrations:

other: on going

None

Conclusions:

On going - Non audited results clearly tends to negative results

Executive summary:

On going - Non audited results clearly tends to negative results

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Two key study was identified for the genetic toxicity assesment (mutagenicity potential).

Gene mutation assay:

A reverse gene mutation assay performed according to the OECD test guideline No. 471 and the EU Method B.13/14 and in compliance with GLP, several Salmonella typhimurium strains were treated with the test item using the direct plate incorporation method or the pre-incubation method.

A preliminary toxicity test was performed to define the dose levels of Dimethyl Pimelate, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in a total of three independent experiments. The two first experiments were performed with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. A third experiment was then performed with S9 mix.

The preliminary test, both experiments without S9 mix and the first experiment with S9 mix were performed according to the direct plate incorporation method. The second experiment with S9 mix was performed according to the pre-incubation method (i.e.60 minutes). The third experiment with S9 mix was performed both according to the direct plate incorporation method and pre-incubation method for comparative purposes, at the request of the Sponsor. 

In the two first experiments, five strains of bacteriaSalmonella typhimuriumwere used: TA 1535, TA 1537, TA 98, TA 100 and TA 102, whereas only the TA 100 strain was used in the third experiment (with S9 mix). Each strain was exposed to at least five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Since the test item was found freely soluble in the final treatment medium and non-toxic in the preliminary test, the highest dose level selected for the main experiments was 5000 µg/plate, according to the criteria specified in the international guidelines.

The mean number of revertants for the vehicle and positive controls met the acceptance criteria. Also, there were at least five analyzable dose levels for each strain and test condition. The study was therefore considered to be valid. No precipitate was observed in the Petri plates when scoring the revertants at any dose levels, either with or without S9 mix.

 

Experiments without S9 mix:

The selected dose levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for the five strains, in both experiments. No noteworthy toxicity was noted at any dose levels, in any strains, in either experiment.

The test item did not induce any noteworthy increase in the number of revertants at any dose levels, in any of the five strains, in either experiment. These results met thus the criteria of a negative response.

 

Experiments with S9 mix:

A moderate toxicity (thinning of the bacterial lawn) was noted at 5000 µg/plate in the TA 1535, TA 98 and TA 102 strains using the pre-incubation method (i.e.in the second experiment). No other noteworthy toxicity was noted in any other strains or test conditions.

- In the first experiment using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels, in any of the five strains.

- In the second experimentusing the pre-incubation method,noteworthy increases in the number of revertants were observed in the TA 98 and TA 100 strains. These increases exceeded the positive threshold of 2-fold the vehicle control value at 5000 µg/plate in the TA 98 strain (3.1-fold the vehicle control value) and at dose levels ≥ 625 µg/plate in the TA 100 strain (up to 6.6-fold the vehicle control value) with an evidence of a dose-response relationship. Moreover,the corresponding mean numbers of revertantswere abovethe vehicle control historical range.

- A third confirmatory experiment was performed both according to the direct plate incorporation method and pre-incubation method for comparative purposes in the TA 100 strain only. 

Using the direct plate incorporation method, the test item did not induce any noteworthy increase in the number of revertants at any dose levels in the TA 100 strain.

Using the pre-incubation method, noteworthy increases in the number of revertants were again observed in the TA 100 strain. These increases exceeded the positive threshold of 2-fold the vehicle control value at dose levels ≥ 625 µg/plate (up to 4.8-fold the vehicle control value) with an evidence of a dose‑response relationship. Moreover, the corresponding mean numbers of revertantswere abovethe vehicle control historical range.

 

Since the results obtained with the TA 100 strain in the first and second experiments with S9 mix were reproduced in this third experiment,they were considered to be biologically relevant, and to show a clear positive response using the pre-incubation method. As a consequence,checking for reproducibility of the increase observed in the TA 98 strain in the second experiment with S9 mix was considered unneedful andno additional experiment was undertaken.

Under the experimental conditions of this study, the test item Dimethyl Pimelate showed a mutagenic activity in the presence of a rat liver metabolizing system using the pre-incubation method inthe bacterial reverse mutation test withSalmonella typhimuriumstrains.

MLA Assay:

Non audited results clearly tends to negative results. the study is on going

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, an additional classification is not required regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the UN-GHS.