Dihexyl fumarate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: Negative (with and without metabolic activation)

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Mouse Lymphoma Assay: Negative (with and without metabolic activation)

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Chromosome aberration test: Positive (without metabolic activation)

Response due to mainly break-type aberrations may be induced by a cytotoxic mechanism rather than a genotoxic mechanism and, therefore, have reduced biological relevance. In the MLA study however, no genotoxic activity was recorded in the any of the exposure conditions. When an MLA study is performed the test cultures undergo a 48-hour expression period prior to plating out for viability and expression of mutagenicity. During this expression period any mutagenic response that induces high levels of toxicity in the cells results in cell death due to lowered viability. In the chromosome aberration data the lack of an expression period results in the presence of mutagenic events that would most likely be non-viable if the cultures were maintained for a greater period of time.

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:

Experimental Starting Date: 7th of July 2004. Experimental Completion Date: 19th of July 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

pre-incubation assay performed in 50 µL of test solution instead of 100µL. Reason: In the pre-incubation test higher concentrations of ethanol can lead to irregular structures in the overlay agar and thus interfere with the counting of the colonies.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identity: DIHEXYL FUMARATE
Batch No.: 9000559308
Aggregate State at Room Temperature:
Colour: liquid colourless
Purity: 98.3%
Stability in solvent: not indicated by the sponsor
Storage: room temperature, light protected
Expiration Date: May 03, 2006

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The concentration range included two logarithmic decades. The following concentrations were tested:
10; 33; 100; 333; 1000; 2500, and 5000 μg/plate

Vehicle / solvent:

On the day of the experiment, the test item DIHEXYL FUMARATE was dissolved in ethanol (purity> 99 %, MERCK, D-64293 Darmstadt). The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, 4-NOPD // 2-aminoanthracene, 2-AA

Details on test system and experimental conditions:

Characterisation of the Salmonella typhimurium Strains
The histidine dependent strains are derived from S. typhimurium strain LT2 through a mutation in the histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty lipopolysaccharide envelope which enables substances to penetrate the cell wall more easily. A further mutation (deletion of the uvrB gene) causes an inactivation of the excision repair system. The latter alteration also includes a deletion in the nitrate reductase and biotin genes. In the strains TA 98, TA 100 and TA 102 the R-factor plasmid pKM 101 carries umu DC analogous genes that are involved in error-prone repair and the ampicillin resistance marker. The strain TA 102 does not contain the uvrS--mutation and is excision repair proficient. Additionally, TA 102 contains the multicopy plasmid pAQ1 carrying the hisG428 mutation ( ochre mutation in the hisG gene ) and a tetracycline resistance gene.

In summary, the mutations of the TA strains used in this study can be described as follows:
Salmonella typhimurium
Strains // Genotype // Type of mutations indicated
TA 1537 // his C 3076; rfa-; uvrB-: // frame shift mutations
TA98 // his D 3052; rfa-; uvrB-;R-factor // frame shift mutations
TA 1535 // his G 46; rfa-; uvrS-: // base-pair substitutions
TA 102 // his G 428; rfa-; uvrB+;R-factor // base-pair substitutions
TA 100 // his G 46; rfa-; uvrB-;R-factor // base-pair substitutions

Regular checking of the properties of the strains regarding the membrane permeability, ampicillin- and tetracycline resistance as well as spontaneous mutation rates is performed in the laboratory of RCC Cytotest Cell Research according to B. Ames et al. and D. Maron and B. Ames. In this way it was ensured that the experimental conditions set down by Ames were fulfilled.
The bacterial strains TA 1535, TA 1537 TA 98, TA 100 and TA 102 were obtained from Trinova Biochem GmbH (35394 Gießen, Germany).

Storage
The strain cultures were stored as stock cultures in ampoules with nutrient broth + 5 % DMSO (MERCK, D-64293 Darmstadt) in liquid nitrogen.

Precultures
From the thawed ampoules of the strains 0.5 ml bacterial suspension was transferred into 250 ml Erlenmeyer flasks containing 20 ml nutrient medium. A solution of 20 µl ampicillin (25 µg/ml) was added to the strains TA 98, TA 100, and TA 102. Additionally 20 µl tetracycline (2 µg/ml) was added to strain TA 102. This nutrient medium contains per litre:
8 g Merck Nutrient Broth (MERCK, D-64293 Darmstadt)
5 g NaCl (MERCK, D-64293 Darmstadt)

The bacterial cultures were incubated in a shaking water bath for 4 hours at 37° C.

Selective Agar
The plates with the minimal agar were obtained from E. Merck, D-64293 Darmstadt.

Overlay Agar
The overlay agar contains per litre:
6.0 g MERCK Agar Agar*
6.0 g NaCl*
10.5mg L-Histidine x HCl x H2O *
12.2 mg Biotin*
* (MERCK, D-64293 Darmstadt)

Sterilisations were performed at 121° C in an autoclave.

Rationale for test conditions:

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in this pre­ experiment were the same as described for the experiment I below (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as f experiment I since the criteria mentioned above were met. Based on the toxic effects observed in strain TA 102 seven concentrations were tested in both main experiments.

Evaluation criteria:

Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate with s9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. II, at 5000 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I, at 1000 - 5000 µg/plate with S9, and Exp. II, at 2500, 5000 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

DISCUSSION OF RESULTS

The test item DIHEXYL FUMARATE was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

 

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls, were tested in triplicate. The test item was tested at the following concentrations:

 

10; 33; 100; 333; 1000; 2500, and 5000 µg/plate

 

In both experiments, reduced background growth was observed with and without S9 mix at higher concentrations in all strains used.

 

Toxic effects, evident as a reduction in the number of revertants (below the factor of 0.5) were observed at the following concentrations (µg/plate):

 

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	/	/	/	5000
TA 1537	/	/	/	5000
TA98	/	/	/	/
TA 100	/	/	/	/
TA 102	/	1000 - 5000	/	2500, 5000

/ no toxic effects observed

 

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with DIHEXYL FUMARATE at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

 

Appropriate reference mutagens were used as positive controls. They showed a distinct in­ crease in induced revertant colonies.

 

In experiment II with metabolic activation, the number of colonies did not quite reach the lower limit of our historical control data in the negative control of strain TA 1535. Since this deviation is rather small, this effect is judged to be based upon biological fluctuations and has no detrimental impact on the outcome of the study.

 

The historical range of positive controls was exceeded in strains 1537 and TA 98 (experiment II) with metabolic activation. This effect indicates the sensitivity of the strains rather than compromising the assay.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 06 March 2018 and 23 April 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Identification: Dihexyl fumarate (EC 242-833-4)
Batch Number: 800317920
CAS Number: 19139-31-2
Purity: >96%
Physical State/Appearance: Clear colorless liquid
Expiry Date: 5 January 2019
Storage Conditions: Room temperature, in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (4%) of the test item.

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human peripheral blood from the following donors: Preliminary Toxicity Test: male, aged 28 years; Main Experiment: male, aged 28 years

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

Metabolic activation:

with and without

Metabolic activation system:

induced rat liver homogenate metabolizing system (S9), at a 2% final concentration

Test concentrations with justification for top dose:

The molecular weight of the test item was given as 284.39, therefore, the maximum dose level was 2000 µg/mL, the maximum recommended dose level. The purity of the test item was 96% and was accounted for in the test item formulations.
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/mL. The maximum dose was the maximum practical concentration.
Main experiment dose levels: See "Any Other Information on methods"

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: The test item was insoluble in DMSO at 200 mg/mL but was soluble in Acetone at 200 mg/mL in solubility checks performed in house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent: Minimal Essential Medium

Positive control substance:

mitomycin C

Remarks:

without metabolic acitivation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

Solvent: DMSO

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 14/12/17 was used in this study. Each batch of S9 is routinely tested for its capability to activate the known mutagens in the Ames test. A copy of the S9 Certificate of Efficacy is presented in Appendix 2.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Test Item Preparation and Analysis
Due to the sensitivity of human lymphocytes to acetone, the formulations were prepared at twice the concentration required in culture and dosed in 50 µL aliquots. Consequently, the maximum practical concentration was 1000 µg/mL.
Prior to each experiment, the test item was accurately weighed, formulated in Acetone and appropriate serial dilutions prepared.
The solubility of the test item was investigated in the Envigo Research Limited Mouse Lymphoma assay, Study number DL14JV.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).
 
The pH and osmolality readings are presented in the following table:
Dose level (µg/mL) 0 3.91 7.81 15.63 31.25 62.5 125 250 500 1000
pH 7.24 7.18 7.22 7.24 7.27 7.27 7.28 7.28 7.30 7.32
Osmolality (mOsm) 381 383 379 380 - 371 - 377 375 358
- = Not performed for this dose concentration
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9)
As the exposure was continuous, the cultures were established, at a nominal volume of 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 3.91 to 1000 µg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

Main Experiment
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 4 to 128 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 4 to 128 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 2 to 64 µg/mL.

Cell Harvest
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) 2.5 hours (tox) and two hours (main) before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding
The slides were coded using a computerized random number generator. Supplementary slides were coded manually.

Mitotic Index
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

Scoring of Chromosome Damage
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.

Evaluation criteria:

The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.

Key result

Species / strain:

lymphocytes: Human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/mL. The maximum dose was the maximum practical concentration.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 125 µg/mL, in the 4(20)-hour in the absence of S9 and at and above 62.5 µg/mL in the 4(20) in the presence of S9 and the continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24 hour continuous exposure was 62.5 µg/mL. The test item induced some evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 128 µg/mL for the 4(20)-hour exposure groups and was 64 µg/mL for the continuous exposure group.

Chromosome Aberration Test – Main Experiment
The qualitative assessment of the slides determined that there was precipitate and the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present in all three exposure groups. In the 4(20)-hour absence and presence of metabolic activation (S9), the maximum dose level of the test item with metaphases suitable for scoring was 128 µg/mL. In the continuous exposure the maximum dose level of the test item with metaphases suitable for scoring was 64 µg/mL.
Precipitate observations were made at the end of exposure in blood-free cultures and was noted at 128 µg/mL, in the 4(20)-hour exposure groups in the absence and presence of S9, and at 64 µg/mL, in the 24-hour continuous exposure group.
They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 11% mitotic inhibition was achieved at 128 µg/mL. In the presence of S9, no dose-related inhibition of mitotic index was observed. An inhibition of mitotic index of 36 and 45% was noted respectively at 48 and 64 µg/mL in the 24-hour continuous exposure group.
The maximum dose level selected for metaphase analysis was the lowest precipitating dose level.
The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.
All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.
The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.
The required number of cells and concentrations were analyzed.
The test item induced statistically significant increases in the frequency of cells with aberrations in the 24 hour continuous exposure group in the absence of metabolic activation.
The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

Mitotic Index - Preliminary Toxicity Test

Dose Level (µg/mL)	4(20)-Hour Without S9		4(20)-Hour With S9		24-Hour Without S9
	Mitotic Index	% of Control	Mitotic Index	% of Control	Mitotic Index	% of Control
0	12.60	100	8.15	100	8.65	100
3.91	-	-	-	-	-	-
7.81	-	-	-	-	9.35	108
15.63	-	-	-	-	4.40	51
31.25	11.35	90	9.60	118	6.00	69
62.5	6.90	55	10.50 P	129	3.70 P	43
125	7.25 P	58	5.85 P	72	- P	-
250	8.90 P	71	- P	-	- P	-
500	- P	-	- P	-	- P	-
1000	- P	-	- P	-	- P	-

- = Not assessed for mitotic index

P = Precipitate observed at end of exposure period in blood-free cultures

Mitotic Index – Main Experiment (4(20)-hour Exposure Groups)

Dose Level (mg/mL)	4(20)-Hour Without S9				4(20)-Hour With S9
	A	B	Mean	% of Control	A	B	Mean	% of Control
0	5.50	5.05	5.28	100	3.75	3.90	3.82	100
4	-	-	-	-	-	-	-	-
8	-	-	-	-	-	-	-	-
16	5.85	5.45	5.65	107	3.70	4.85	4.27	112
32	4.75	5.25	5.00	95	3.85	6.70	5.28	138
64	4.20	4.80	4.50	85	3.80	3.65	3.73	98
128	4.90 P	4.50 P	4.70 P	89	4.60 P	3.85 P	4.23 P	111
MMC 0.2	2.20	1.50	1.85	35	NA	NA	NA	NA
CP 2	NA	NA	NA	NA	2.55	1.40	1.98	52

MMC           =Mitomycin C

CP                =Cyclophosphamide

P                   =Precipitateat end of exposure period in blood-free cultures

NA               =Not applicable

-                    =Not assessed for mitotic index

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level (µg/mL)	24-Hour Without S9
	A	B	Mean	% of Control
0	6.50	7.55	7.03	100
2	-	-	-	-
4	-	-	-	-
8	-	-	-	-
16	6.15	6.15	6.15	87
32	5.30	6.75	6.03	86
48	4.25	4.75	4.50	64
64	4.00 P	3.80 P	3.90 P	55
MMC 0.1	3.65	3.00	3.33	47

MMC = Mitomycin C

P  = Precipitateat end of exposure period in blood-free cultures

-  = Not assessed for mitotic index

Mean Frequency of Polyploid Cells (%)

Main Experiment

Dose Level (µg/mL)	Exposure Group
	4(20)-Hour Without S9	4(20)-Hour With S9	24-Hour Without S9
0	0	0	0
2	NA	NA	-
4	-	-	-
8	-	-	0
16	0	0	0
32	0	0	0
64	0	0	0
128	0	0	NA
MMC 0.2	0	NA	NA
MMC 0.1	NA	NA	0
CP 2	NA	0	NA

MMC    Mitomycin C

CP        Cyclophosphamide

NA       Not applicable

Conclusions:

Dihexyl fumarate (EC 242-833-4) induced a statistically significant increase in the frequency of cells with chromosome aberrations, in the absence of a liver enzyme metabolizing system following 24-hour exposure. The test item was, therefore, considered to be clastogenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of anin vitrostudy for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Experiment were as follows:

Group	Final concentration of test itemDihexylfumarate (EC 242-833-4)(µg/mL)
4(20)-hour without S9	0, 4, 8, 16, 32, 64, 128
4(20)-hour with S9 (2%)	0, 4, 8, 16, 32, 64, 128
24-hour without S9	0, 2, 4, 8, 16, 32, 48, 64

Results

All vehicle (Acetone) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item induced statistically significant increases in the frequency of cells with aberrations in the 24 hour continuous exposure group, using a dose range that included a dose level that was approaching optimum toxicity. However, it should be noted that the response was solely due to break-type aberrations which are often a result of a cytotoxic mechanism rather than a mutagenic mechanism.

Conclusion

The test item, Dihexyl fumarate (EC 242-833-4)was considered to be clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date:19 February 2018, Experimental completion date: 13 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

Identification: Dihexyl fumarate (EC 242-833-4)
Batch Number: 800317920
CAS Number: 19139-31-2
Purity: >96%
Molecular Formula: C16H28O4
Molecular Weight: 284.39
Physical State/Appearance: Clear colorless liquid
Expiry Date: 25 January 2019
Storage Conditions: Room temperature, in the dark
A correction for the purity of the test item was applied to the formulations.

Target gene:

The thymidine kinase heterozygote system, TK +/- to TK -/-. Based upon the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (2.5 to 60 µg/mL in the 4-hour exposure group in the absence of metabolic activation, 1.95 to 250 µg/mL in the 4 hour exposure group in the presence of metabolic activation, and 1.25 to 30 µg/mL in the 24-hour exposure group in the absence of metabolic activation), vehicle and positive controls.

4-hour without S9: 10, 20, 30, 40, 50, 60 µg/mL
4-hour with S9 (2%): 3.91, 7.81, 15.63, 31.25, 62.5, 125 µg/mL
24-hour without S9: 5, 10, 15, 20, 25, 30 µg/mL

Vehicle / solvent:

acetone

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 0.98 to 250 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose (mg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
0.98	98	97	98
1.95	89	91	106
3.91	87	94	121
7.81	89	95	85
15.63	70	100	36
31.25	39	85	10
62.5	5	76	0
125	7	67	0
250	37	72	0

There was evidence of modest dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour exposure group in the presence of metabolic activation, and marked reductions in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Precipitate of the test item was observed at and above 125 µg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and at 250 µg/mL in the 4-hour exposure group in the presence of metabolic activation, at the end of the exposure periods. Therefore,following the recommendations of the OECD 490 guideline, the maximum dose levels in the Mutagenicity Test were limited by the onset of test item precipitate in the 4-hour exposure group in the presence of metabolic activation, and test item‑induced toxicity in the 4-hour and 24-hour exposure groups in the absence of metabolic activation.

 

Mutagenicity Test

 

Concentration	4-Hours-S9			Concentration	4-Hours+S9			Concentration	24-Hours-S9
(µg/mL)	%RSG	RTG	MF§	(µg/mL)	%RSG	RTG	MF§	(µg/mL)	%RSG	RTG	MF§
0	100	1	137.68	0	100	1	153.14	0	100	1	168.76
2.5 Ø	96			1.95 Ø	102			1.25 Ø	91
5 Ø	95			3.91	97	0.98	151.92	2.5 Ø	100
10	89	0.98	149.12	7.81	103	1.06	139.28	5	87	0.93	157.93
20	77	0.83	166.75	15.63	89	0.92	122.27	10	61	0.81	179.41
30	62	0.57	181	31.25	96	0.87	155.44	15	47	0.75	156.12
40	52	0.51	155.31	62.5	88	0.79	171.74	20	37	0.61	165.8
50	28	0.32	154.75	125	64	0.57	166	25	27	0.53	156.13
60	19	0.19	175.18	250 Ø	57			30	17	0.26	194.19
MF threshold for a positive response = 263.68				MF threshold for a positive response = 279.14				MF threshold for a positive response = 294.76
Positive control				Positive control				Positive control
EMS				CP				EMS
400	73	0.63	1085.12	1.5	71	0.41	1228.33	150	54	0.49	1588.13

%RSG=         Relative Suspension Growth

RTG   =         Relative Total Growth

Ø = Not plated, surplus to requirements

CP = Cyclophosphamide

EMS = Ethylmethanesulphonate

MF§ = 5-TFT resistant mutants/106 viable cells 2 days after exposure

As was seen previously, there was evidence of modest dose-related toxicity in cells treated with the test item in the 4-hour exposure group in the presence of metabolic activation, and marked reductions in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, as indicated by the %RSG and RTG values. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved in the 4-hour and 24-hour exposure groups in the absence of metabolic activation (Tables 3 and 9). There was evidence of a modest reduction in viability (%V) in the 24-hour exposure group in the absence of metabolic activation, indicating that modest residual toxicity had occurred (Table 9). At the end of the exposure period, precipitate of the test item was observed at 125 and 250 µg/mL in the 4-hour exposure group in the presence of metabolic activation. Therefore, following the recommendations of the OECD 490 guideline, the lowest precipitating dose level of 125 µg/ mL was plated for viability and 5‑TFT resistance. Acceptable levels of toxicity were seen with the positive control substances.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6per viable cell at any of the dose levels, including the dose levels that achieved optimum toxicity in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and the lowest precipitating dose level in the 4-hour exposure group in the presence of metabolic activation.

 

 

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

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

In the Ames study, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Furthermore, in the MLA study, the test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic.

The chromosome aberration study was considered positive due to clastogenic effects. It was summised that these may have been as a result of a cytotoxic mechanism rather than a genotoxic mechanism. As such classification was determiend based on the other two studies to be negative.