Dibenzylbenzene, ar-methyl derivative

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two recent GLP reliable studies (Ames test, in vitro mamalian cell gene mutation test, 2019) showing no alert on mutagenicity.

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:

15 March 2019 - 09 April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21st july 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

n/a

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

The selected dose levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at required concentrations.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-anthramine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation). 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.

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Merhod: observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Rationale for test conditions:

Not applicable.

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.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Emulsion

RANGE-FINDING STUDY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 500 µg/plate. This emulsion prevented the scoring of the revertants at 5000 µg/plate in the TA 98 strain.
No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards the three strains used, either with or without S9 mix.

RESULTS OF CYTOTOXICITY and GENOTOXICITY:
A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 250 µg/plate.
No noteworthy toxicity was noted at any dose levels, towards the five strains used.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%): see attached

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium strains, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14).  A preliminary toxicity test was performed to define the dose levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both 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.

 

Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, at 37°C).

 

Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, 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.

 

Results

The test item was found non-toxic in the preliminary test, but poorly soluble in the final treatment medium (emulsion in the Petri plates). Consequently, the selection of the highest dose level to be used in the main experiments was based on the level of emulsion, 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 five analyzable dose levels for each strain and test condition. The study was therefore considered to be valid.

 

The selected dose levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.

 

A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 250 µg/plate.

 

No noteworthy toxicity was noted at any dose levels, towards the five strains used.

 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment, with or without S9 mix. These results met the criteria of a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or absence of a rat liver metabolizing system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 March 2019 - 26 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

n/a

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Vehicle / solvent:

- Vehicle used: dimethylsulfoxide.
- Justification for choice: using a test item concentration of 500 mg/mL and a treatment volume of 1% (v/v) in the culture medium (i.e. 200 µL/20 mL culture medium), the highest recommended dose level of 5000 µg/mL was achievable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE2).

Rationale for test conditions:

Since the test item was found cytotoxic in the preliminary test and poorly soluble in the culture medium at the end of treatment, the selection of the highest dose level for the main experiment was based both on the level of precipitate (emulsion) and cytotoxicity, according to the criteria specified in the international guidelines.

Evaluation criteria:

In all cases, biological relevance was taken into consideration when evaluating the results.

Evaluation of a positive response:
Based on IWGT recommendations, 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%, are not 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:
- 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.

Statistics:

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

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See § Summary

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: at the end of the treatment periods, an emulsion was observed in the culture medium at dose levels = 12.5 µg/mL.

RANGE-FINDING STUDIES:
Using a test item concentration of 500 mg/mL and a treatment volume of 1% (v/v) in the culture medium (i.e. 200 µL/20 mL culture medium), the highest recommended dose level of 5000 µg/mL was achievable. Thus the dose levels selected for the treatments of the preliminary test were 8.19, 20.5, 51.2, 128, 320, 800, 2000 and 5000 µg/mL.

At the highest tested dose level, i.e. 5000 µg/mL, the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality was 386 mOsm/kg H2O (452 mOsm/kg H2O for the vehicle control). Thus none of the selected dose levels was considered to produce extreme culture conditions.

At the end of the treatment periods, an emulsion was observed in the culture medium at dose levels = 20.5 µg/mL.

Without S9 mix, a severe cytotoxicity was observed at dose levels = 51.2 µg/mL, as shown by a 100% decrease in the Adj. RTG.
With S9 mix, a moderate to severe cytotoxicity was induced at dose levels = 20.5 µg/mL, as shown by a 40 to 100% decrease in the Adj. RTG.

Cytotoxicity
A moderate to severe cytotoxicity was induced at dose levels = 37.5 µg/mL, as shown by a 70 to 96% decrease in Adj. RTG.

Mutagenicity
No noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the dose levels inducing acceptable level of cytotoxicity, i.e. up to 37.5 µg/mL, while insolubility was observed in the culture medium from the dose level of 12.5 µg/mL. No dose-response relationship was demonstrated by the linear regression (p > 0.05). These results met the criteria for a negative response.

Conclusions:

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells.

The study was performed according to international guidelines (OECD No. 490 and Council Regulation (EC) No. 440/2008 of 30 May 2008) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in a single mutagenicity experiment, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 10⁵cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a, 5% CO₂humidified incubator.

Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

Results

Since the test item was found cytotoxic in the preliminary test and poorly soluble in the culture medium at the end of treatment, the selection of the highest dose level for the main experiment was based both on the level of precipitate (emulsion) and cytotoxicity, according to the criteria specified in the international guidelines.

 

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria.In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid.

 

The selected dose levels were:

.  1.56, 3.13, 6.25, 12.5, 18.8, 25, 37.5 and 50 µg/mL for the 3-hour treatment without S9 mix,

.   1.56, 3.13, 6.25, 12.5, 25, 50, 75 and 100 µg/mL for the 3-hour treatment with S9 mix.

 

At the end of the treatment, an emulsion, which did not prevent any scoring, was observed in the culture medium at dose levels = 12.5 µg/mL.

 

Cytotoxicity

Without S9 mix, a moderate to severe cytotoxicity was induced at dose levels = 37.5 µg/mL, as shown by a 70 to 96% decrease in Adj. RTG.

With S9 mix, a moderate to marked cytotoxicity was induced at dose levels = 50 µg/mL, as shown by a 41 to 79% decrease in the Adj. RTG.

 

Mutagenicity

Without S9 mix, no noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the dose levels inducing acceptable level of cytotoxicity, i.e. up to 37.5 µg/mL, while insolubility was observed in the culture medium from the dose level of 12.5 µg/mL. No dose-response relationship was demonstrated by the linear regression (p > 0.05).

 

With S9 mix, no noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression (p > 0.05).

 

These results met the criteria for a negative response.

 

Conclusion

Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There is one in GLP vivo micronucleus test (OECD 474, Sanofi, 1990) showing no alert in vivo.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From December 13, 1989 to April 20, 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

- Test was performed on males only instead of males and females, without any justification, - 5 rats were used but one gave a slide with insufficient cell density, leading to conclude with only 4 animals, - Positive control was not concurrently tested.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Study conducted prior to the adoption of the most

Deviations:

yes

Remarks:

Animals were treated for 4 months; only one dose was tested: no cytotoxicity was elicited at this only tested dose level; only male animals were used; 1000 PCE were scored for micronuclei instead of at least 2000. 5 rats were used but one gave a slide wi

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, France
- Age at study initiation: approximately 7 weeks old
- Weight at study initiation: Mean initial bodyweight for males was 185 g
- Fasting period before study: not reported
- Housing: individually; space allocated: 345 square cm x 17 cm
- Diet: a complete commercial diet from U..A.R, ad libitum
- Water: tap-water through automatic waterers, ad libitum
- Acclimation period: at least 1 week before beginning treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 60 ± 10
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 August To: 13 December 1989

Route of administration:

oral: gavage

Vehicle:

10% aqueous gum arabic solution

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
They were prepared just before administration as suspensions in 10% aqueous gum arabic solution.

Duration of treatment / exposure:

At least 120 days

Frequency of treatment:

daily

Post exposure period:

no

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control test was designed for the validation of the purification method, but not for the validation of the specific assay with test substance. Cyclophosphamide was administred intraperitoneally as a single dose at 15 mg/kg bw. Test with positive control was conducted separately from the main study with test substance, but at the same period, i.e. January 3, 1990.
- Justification for choice of positive control(s): no
- Route of administration: intraperitoneally
- Doses / concentrations: 15 mg/kg bw

Tissues and cell types examined:

Smears of bone marrow of each femur were prepared. Erythrocytes were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The in vivo genotoxic potential of Jarylec DBT was assessed in the male rat as part of a 4-month toxicity study

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Necropsy was performed from Day 134 to 143 (final kill, all surviving animais).

DETAILS OF SLIDE PREPARATION:
Rat bone marrow slides were prepared after purification and enrichment of polychromatic erythrocyte fractions according to Romagna and Staniforth's method (1989).
At the scheduled sacrifice times (from days 134 to 143), five mice per treatment were anaesthetized by pentobarbital and exsanguinated.
Immediately following sacrifice, the femurs were removed and the bone marrow was flushed with newborn calf serum (NCS) supplemented with 25 mM EDTA. The bone marrow cell suspension was purified in a column filled with a mixture of microcrystalline cellulose and alpha-cellulose fibres (50/50).
Elution was performed using 25 ml HBSS (calcium- and magnesium-free). The eluate was filtered at the column exit and centrifuged for 10 min
at 1700 rpm. The supernatant was drawn off and the remaining cell pellet was resuspended in 500 µl NCS. A step-gradient separation was performed using Percoll solutions (at 80% and 30%). After centrifugation for 10 min at 1700 rpm, the sharp band
at the interface of the 30/80% gradient was collected and diluted with HBSS. After an additional centrifugation at 1500 rpm for 5 minutes, the supernatant was discarded and cells were resuspended in 500 µl NCS. Purified cell suspension were diluted in NCS (1:28 or 1:40) and 50 µl were spread onto clean glass slides. The slides were stained with Wright using an automatic Hemostain equipment (Geometric Data Corporation). - PCE/NCE ratio was determined until a total of at least 1000 cells (PCE+NCE) were counted - A total of 1000 PCE was examined for micronuclei.

METHOD OF ANALYSIS:
Reading was performed using a microscope (immersion lens x 100). Evaluation of bone marrow differential cell counts involved:
- the number of micronucleated polychromatic cells per 1000 polychromatic erythrocytes,
- the number of micronucleated normochromatic cells during the readings;
- the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes. Both cellular types were counted until the first 1000 PCE were numbered.
- the increase in micronucleated polychromatic cells indicates a possible genotoxic effect whereas the decrease in PCE/NCE ratio shows medullary toxicity.

Evaluation criteria:

Criteria for recognizing the micronuclei are given below:
- shape and color giving them the aspect of small nuclei, with well defined outlines, 1/20th to 1/50th the size of an erythrocyte;
- structure identical to that of a nucleus (no refraction at focus);
- difference from an artefact which is an element appearing indifferently in all types of cells, and sometimes outside cells.

Statistics:

The mean and standard error of the mean were calculated, and treated groups were compared with the control group using the Kruskal-Wallis non parametric test.When the test was significant (non homogeneous means), this method was used for group comparisons; when not, means were considered as homogeneous.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY (see table 1 in the field Remarks on results)
- Induction of micronuclei (for Micronucleus assay): no increase in the number of micronucleated, normochromatic or polychromatic cells was noted in animals treated with Jarylec DBT at 500 mg/kg/d.
- Ratio of PCE/NCE (for Micronucleus assay): no significant decrease in the PCE/NCE ratio was noted, indicating the absence of medullary toxicity of Jarylec DBT.

Conclusions:

Dibenzylbenzene, ar-methyl derivative did not induce any increase in the proportion of micronuclei per cell after a 4-month treatment in the rat at 500 mg/kg bw/day by gavage.

Executive summary:

As part of the 4-month toxicity study (see section 7.5.1 Repeated dose toxicity oral) with Dibenzylbenzene, ar-methyl derivative (JARYLEC DBT) administered orally by gavage (0, 5, 50, and 500 mg/kg/day) to Sprague-Dawley rats, bone marrow slides were collected in male animals to evaluate the number of micronuclei per 1000 polychromatic erythrocytes. Finally, only slides of control and high dose treated groups were read.

Bone marrow slides were prepared from 5 male rats/group after purification and enrichment of polychromatic fractions on Percoll gradient. Scoring of the number of micronucleated polychromatic cells per 1000 polychromatic erythrocytes, the number of micronucleated normochromatic cells during the reading, and the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes was performed on control and high dose treated groups.

No increase in the number of micronucleated, normochromatic or polychromatic cells, and no significant decrease in the PCE/NCE ratio were noted in rats treated at 500 mg/kg bw/d for approximately 4 months.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

The mutagenic properties of Dibenzylbenzene, ar-methyl derivative have been evaluated in various studies, both in vitro and in vivo. In all available studies no mutagenicity was observed and it therefore can be concluded that Dibenzylbenzene, ar-methyl derivative is not mutagenic.

in vitro gene mutation study in bacteria (CIT 2019)

The objective of this study was to evaluate the potential of the test item to induce reverse mutations in Salmonella typhimurium. The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B13/14).  A preliminary toxicity test was performed to define the dose levels of the test item, diluted in dimethylsulfoxide (DMSO), to be used for the mutagenicity experiments. The test item was then tested in two independent experiments, both 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. Treatments were performed according to the direct plate incorporation method except for the second experiment with S9 mix, which was performed according to the pre-incubation method (60 minutes, at 37°C). Five strains of bacteria Salmonella typhimurium were used: TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain was exposed to five dose levels of the test item (three plates/dose level). After 48 to 72 hours of incubation at 37°C, 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.

The test item was found non-toxic in the preliminary test, but poorly soluble in the final treatment medium (emulsion in the Petri plates). Consequently, the selection of the highest dose level to be used in the main experiments was based on the level of emulsion, 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 five analyzable dose levels for each strain and test condition. The study was therefore considered to be valid. The selected dose levels were 62.5, 125, 250, 500 and 1000 µg/plate for the five strains, in both mutagenicity experiments with and without S9 mix.  A moderate to strong emulsion was observed in the Petri plates when scoring the revertants at dose levels >= 250 µg/plate. No noteworthy toxicity was noted at any dose levels, towards the five strains used. 

The test item did not induce any noteworthy increase in the number of revertants, in any of the five tested strains, in either experiment, with or without S9 mix. These results met the criteria of a negative response. Under the experimental conditions of this study, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or absence of a rat liver metabolizing system.

 

In vitro gene mutation study (CIT 2019b)

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/- mouse lymphoma cells. The study was performed according to international guidelines (OECD No. 490 and Council Regulation (EC) No. 440/2008 of 30 May 2008) and in compliance with the principles of Good Laboratory Practices.

After a preliminary cytotoxicity test, the test item diluted in dimethylsulfoxide (DMSO), was tested in a single mutagenicity experiment, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Cultures of 20 mL at 5 x 10^5 cells/mL were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% in a, 5% CO2 humidified incubator. Cytotoxicity wasmeasured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) and Cloning Efficiency following the expression time (CE₂). The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

Since the test item was found cytotoxic in the preliminary test and poorly soluble in the culture medium at the end of treatment, the selection of the highest dose level for the main experiment was based both on the level of precipitate (emulsion) and cytotoxicity, according to the criteria specified in the international guidelines.

 

The cloning efficiencies, the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria.

For the positive control cultures, the increase in the mutation frequencies met also the acceptance criteria.In addition, the upper limit of cytotoxicity observed in the positive control cultures had an Adj. RTG greater than 10%.The study was therefore considered to be valid. At the end of the treatment, an emulsion, which did not prevent any scoring, was observed in the culture medium at dose levels = 12.5 µg/mL.

Without S9 mix, a moderate to severe cytotoxicity was induced at dose levels = 37.5 µg/mL, as shown by a 70 to 96% decrease in Adj. RTG. Without S9 mix, no noteworthy increase in the mutation frequency was noted relative to the corresponding vehicle control, at any of the dose levels inducing acceptable level of cytotoxicity, i.e. up to 37.5 µg/mL, while insolubility was observed in the culture medium from the dose level of 12.5 µg/mL. No dose-response relationship was demonstrated by the linear regression (p > 0.05).

With S9 mix, a moderate to marked cytotoxicity was induced at dose levels = 50 µg/mL, as shown by a 41 to 79% decrease in the Adj. RTG. With S9 mix, no noteworthy increase in the mutation frequency was noted at any of the tested dose levels, relative to the corresponding vehicle control, and no dose-response relationship was demonstrated by the linear regression (p > 0.05).

These results met the criteria for a negative response. Under the experimental conditions of this study, the test item did not show any mutagenic activity in the mouse lymphoma assay, either in the presence or absence of a rat liver metabolizing system.

 

In vivo micronucleus assay (1990)

As part of the 4-month toxicity study (see section 7.5.1 Repeated dose toxicity oral) with Dibenzylbenzene, ar-methyl derivative (JARYLEC DBT) administered orally by gavage (0, 5, 50, and 500 mg/kg/day) to Sprague-Dawley rats, bone marrow slides were collected in male animals to evaluate the number of micronuclei per 1000 polychromatic erythrocytes. Finally, only slides of control and high dose treated groups were read.

Bone marrow slides were prepared from 5 male rats/group after purification and enrichment of polychromatic fractions on Percoll gradient. Scoring of the number of micronucleated polychromatic cells per 1000 polychromatic erythrocytes, the number of micronucleated normochromatic cells during the reading, and the ratio of polychromatic (PCE) to normochromatic (NCE) erythrocytes was performed on control and high dose treated groups.

No increase in the number of micronucleated, normochromatic or polychromatic cells, and no significant decrease in the PCE/NCE ratio were noted in rats treated at 500 mg/kg bw/d for approximately 4 months.

Justification for classification or non-classification

No classification for mutagenic effects is indicated according to the classification, labeling and packaging (CLP) regulation (EC 1272/2008).