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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1994-10-18 until 1994-10-25 (plate incorporation test) and 1994-12-14 until 1994-12-20 (preincubation test)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
not specified
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
S. typhimurium TA 1538
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix
Test concentrations with justification for top dose:
8 / 40 / 200 / 1000 / 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: not mentioned
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Nitrofluorene (2.5 µg/plate) for the strains TA 98, 1538; Sodium azide (2.5 µg/plate) for TA 100, 1535; Aminoacredine (25 µg/plate) for TA 1537
Remarks:
without metabolic activation
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Aminoanthracene (2.5 µg/plate) with all strains
Remarks:
with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation, performed in two independent tests


DURATION
- Preincubation period: 30 minutes
- Exposure duration: 96 hours


NUMBER OF REPLICATIONS: 3 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: not mentioned


OTHER EXAMINATIONS:
- Other: Determination of the frequency of induced or spontaneous reversion to histidine independence with negative controls (H2O), solvent controls (DMSO), test substance concentrations and positive controls; determination of the titers of overnight cultures


OTHER: none
Evaluation criteria:
According to Ames a test article which caused no mutagenic effects at a concentration of 5000 µg/plate will be called non-mutagenic.
Statistics:
no statistics performed
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
Species / strain:
S. typhimurium TA 1535
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 1537
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 1538
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
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not applicable
- Evaporation from medium: not applicable
- Water solubility: not mentioned
- Precipitation: at a concentration of 200 µg/plate and higher
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: not performed
COMPARISON WITH HISTORICAL CONTROL DATA: not performed
ADDITIONAL INFORMATION ON CYTOTOXICITY: no signs of cytotoxicity both with and without metabolic activation observed
Remarks on result:
other: strain/cell type: Salmonella typhimurium
Remarks:
Migrated from field 'Test system'.

Table #1: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 98]

[Strain TA 100]

[Strain TA 1535]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

 32±8

 51±4

 no

 117±5

 123±6

 no

 12±5

15±1 

 no

8

 28±1

 60±4

 no

 105±5

 149±3

 no

 7±2

 12±1

 no

40

 34±5

 49±10

 no

 103±4

 146±10

 no

 10±2

 12±6

 no

200

 29±8**

 58±2**

 no

 97±9**

 134±8**

 no

 9±1**

 15±2**

 no

1000

 36±5**

 55±4**

no

 101±16**

 114±3**

 no

 11±6**

 9±2**

 no

5000

 33±4**

 48±7**

 no

 113±9**

 138±21**

 no

 10±1**

 10±3**

 no

Positive control

 204±3

 1657±189

 no

 414±14

 2043±69

 no

 412±29

 191±41

 no

*solvent control with DMSO ** precipitation  

Table #1 continued: Plate incorporation test: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 1537]

[Strain TA 1538]

[-]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)


0*

 16±4

 14±5

 no

 21±4

 33±7

 no

 

 

 

8

 18±3

 16±3

 no

 26±4

 33±5

 no

 

 

 

40

 19±3

 14±6

 no

 26±1

 33±8

 no

 

 

 

200

 18±6**

 16±8**

 no

 32±2**

 35±8**

 no

 

 

 

1000

 20±1**

 19±6**

 no

 25±2**

 31±6**

 no

 

 

 

5000

 17±6**

 20±8**

 no

 22±1**

 34±5**

 no

 

 

 

Positive control

 79±19

 195±60

 no

 239±16

 1839±100

 no

 

 

 

*solvent control with DMSO ** precipitation  

Table #2: Preincubation test: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 98]

[Strain TA 100]

[Strain TA 1535]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

 34±11

 50±4

 no

 136±6

 128±4

 no

 10±3

 14±4

 no

8

 30±4

 50±8

 no

 112±7

 146±22

 no

 9±2

 12±3

 no

40

 37±3

 54±4

 no

 121±4

 138±5

 no

 13±1

 17±0

 no

200

 28±4**

 46±12**

 no

 128±27**

 124±19**

 no

 10±1**

 13±5**

 no

1000

 34±4**

 45±3**

 no

 110±4**

 119±3**

 no

 13±2**

 12±5**

 no

5000

 30±7**

 54±3**

 no

 128±21**

 136±14**

 no

 13±4**

 12±2**

 no

Positive control

 16012

 1945±100

 no

 385±29

 2150±66

 no

 361±10

 217±9

 no

*solvent control with DMSO ** presipitation  

Table #2 continued: Preincubation test: Number of revertants per plate (mean of 3 plates)

 

[Strain TA 1537]

[Strain TA 1538]

[-]

Conc.
[unit]

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)


0*

 37±4

 41±8

 no

 29±6

 37±6

 no

 

 

 

8

 35±3

 41±7

 no

 22±9

 35±8

 no

 

 

 

40

 32±7

 47±12

 no

 22±5

 45±1

 no

 

 

 

200

 29±8**

 41±7**

 no

 20±3**

 36±2**

 no

 

 

 

1000

 35±6**

 46±6**

 no

 18±4**

 32±6**

 no

 

 

 

5000

 28±1**

 45±14**

 no

 24±5**

 38±9**

 no

 

 

 

Positive control

 218±49

 362±10

 no

 175±2

 953±90

 no

 

 

 

*solvent control with DMSO ** precipitation  

Conclusions:
Interpretation of results (migrated information):
negative

The test material was found to be not mutagenic in a standard bacterial reverse mutation assay (Ames test).
Executive summary:

The test material was found to be not mutagenic in a standard bacterial reverse mutation assay (Ames test) in concentrations up to 5000 µg/plate in the presence and absence of metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-06-17 to 1992-10-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: Ham's F-12 supplemented with 10 % foetal bovine and antibiotics, no further details mentioned
- Properly maintained: maintianed at -196°C in liquid nitrogene using 10 % DMSO in culture medium as cryoprotectant
- Periodically checked for Mycoplasma contamination: no data
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: not applicable
Metabolic activation:
with and without
Metabolic activation system:
microsomal enzyme fraction (S9 mix), prepared from the livers of male Sprague-Dawley rats, which received 500 mg/kg b.w. Aroclor 1254 i.p.; Lot. No. Aro. S9/08/06/92D and Aro. S9/27/07/92B, obtained from the British Biological Research Association
Test concentrations with justification for top dose:
19.5 / 39.1 / 78.13 / 156.25 / 312. 5 / 625 / 1250 / 2500 and 5000 µg/mL in the cytotoxicity study
1250 / 2500 and 5000 µg/mL in the main experiments
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: no data
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone, 1 % (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without metabolic activation Migrated to IUCLID6: 0.05 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Acetone, 1 % (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation Migrated to IUCLID6: 5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Without metabolic activation: 12 resp. 20 hours continuous exposure to the test material
With metabolic activation: 4 hours exposure to the test material and S9 mix, then washing and a further 8 resp. 16 hours in treatment-free media prior to cell harvest
Cell harvest: Mitosis was arrested by addition of demecolcine approx. two hours before the required harvest time. After incubation with demecolcine, the cells were trypsinised to detach them from the tissue culture flask and suspended in culture medium. The cells were centrifuged, followed by hyptonic treatment with 0.075 M KCl and fixation in fresh methanol/glacial acid (3:1 v/v).
Preparation of metaphase spreads: The cells in fixative were dropped onto clean, wet microscope slides and left to air dry, followed by staining, rinsing, drying and mounting in Depex mounting medium.



DURATION
- Preincubation period: not applicable
- Exposure duration: 4 h in the presence of S9 mix; 12 resp. 20 h in the absence of S9 mix
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 12 and 20 h


SELECTION AGENT (mutation assays): not applicable
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine (0.1 µg/mL)
STAIN (for cytogenetic assays): 2 % Gurrs Giemsa R66


NUMBER OF REPLICATIONS: two cultures per dose level


NUMBER OF CELLS EVALUATED: 200 metaphases per dose level (100 per culture)


DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (Growth inhibition was estimated by counting the number of cells at the end of the culture period on an electronic cell counter (Coulter) and expressing the cell count as a percentage of the concurrent negative control value)


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes, but included as polyploid cells
- Other: Mitotic index


OTHER: none
Evaluation criteria:
The metaphases were scored according to the simplified system of Savage (1976) recommended in the UKEMS guidelines for mutagenicity testing.
A positive response was recorded for a particular treatment if the % diploid cells with aberrations (gaps excluded) exceeded the maximum historical value (established in the laboratory in many experiments) and gave a statistically significant increase over the concurrent control value. If only the % diploid cells with aberrations (gaps included) exceed historical values then a +/- response was recorded. Positive responses were also recorded if the % cells with aberrations (gaps excluded) was statistically significant greater than the concurrent control level, even if it was below historical levels, but only if there was an indication of a dose response.
Statistics:
The frequency of cells with aberrations (both including and excluding gaps) and the frequency of polyploid cells was compared with the concurrent vehicle control value using Fisher¿s Exact test.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: test substance unsoluble in water
- Precipitation: Marlotherm S formed a visible fine emulsion at dose levels above 19.5 µg/mL and small oil-like globules were observed at 2500 and 5000 µg/mL.
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: A cytotoxicity test was performed on cell cultures using a 4-hour exposure time with metabolic activation followed by an 8 and 16-hour culture period in treatment free media. Treatment without metabolic activation was continuous with cell harvest at 12 hours and 20 hours.
The slides from the preliminary cytotoxicity were later assessed for numbers of cells with aberrations and mitotic index to ensure that there was no clastogenic effect at lower dose levels. The concentrations assessed were as follows: 19.5, 39, 78 and 156.25 µg/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: The negative control cultures gave values of chromosome aberrations within the expected range
. A weak response was seen in the second experiment with metabolic activation (12 hours sampling time). It was not dose-related and did not appear in the 20-hour cultures. The response was within the historical range for this cell line and was therefore considered to be of no toxicological significance.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test substance showed no evidence of a dose-related increase in cell toxicity at any dose level, either with or without S9.

Table #1: Summary of data obtained in chromosomal aberration test #1

    Treatment                Aberrant Cell Frequency   
  Conc. [µg/mL]   S9 Mix  Harvest Time No. of Cells Scored    Including Gaps          Excluding Gaps
           %  Judge  %  Judge
Vehicle Control  1% Acetone  - 12 h 200  6.0  -  2.0  -
Marlotherm S  1250 - 12 h 200  5.5 neg.  1.5  neg.
Marlotherm S  2500 12 h 200  0.5 neg.  0.0  neg.
Marlotherm S 5000  12 h 200  1.5  neg.  0.0  neg.
 Mitomycin C 0.05  12 h 200  13.5 pos.**  11.5  pos.***
Vehicle Control 1% Acetone  + 12 h 200  5.0  -  1.0  -
Marlotherm S 1250 + 12 h 200  7.5  neg.  3.0  neg.
Marlotherm S 2500 + 12 h 200  6.0  neg.  0.5  neg.
Marlotherm S 5000 + 12 h 200  5.5  neg.  2.0  neg.
Cylophosphamide + 12 h 200  13.5 pos.**  7.5  pos.***
Vehicle control   1 % Acetone  -  20 h  200  5.5  -  2.5  -
 Marlotherm S  1250  -  20 h  200  6.0  neg.  3.0  neg.
 Marlotherm S  2500  -  20 h  200  3.0  neg.  2.0  neg.
 Marlotherm S  5000  -  20 h  200  0.5  neg.  0.0  neg.
 Mitomycin C  0.05  -  20 h  200  24.0  pos.***  14.5  pos.***
Vehicle control   1 % Acetone  +  20 h  200  4.5  -  1.5  -
 Marlotherm S  1250  +  20 h  200  6.0  neg.  1.0  neg.
 Marlotherm S  2500  +  20 h  200  4.0  neg.  3.0  neg.
 Marlotherm S  5000  +  20 h  200  7.5  neg.  3.0  neg.
 Cyclophosphamide  5  +  20 h  200  20.5  pos.***  12.5  pos.***

neg. = statistically negative aberration frequency pos. = statistically positive aberration frequency ** = p<0.01 *** = p<0.001

Table #2: Summary of data obtained in chromosomal test #2

    Treatment                Aberrant Cell Frequency   
  Conc. [µg/mL]   S9 Mix  Harvest Time No. of Cells Scored    Including Gaps          Excluding Gaps
           %  Judge  %  Judge
Vehicle Control  1% Acetone  - 12 h 200  1.0  -  0.5  -
Marlotherm S  1250 - 12 h 200  4.0 neg.  3.0  neg.
Marlotherm S  2500 12 h 200  1.0 neg.  1.0  neg.
Marlotherm S 5000  12 h 200  1.0  neg.  1.0  neg.
 Mitomycin C 0.05  12 h 200  16.0 pos.***  9.5  pos.***
Vehicle Control 1% Acetone  + 12 h 200  2.5  -  0.5  -
Marlotherm S 1250 + 12 h 200  7.0 pos.*  4.5  pos.**
Marlotherm S 2500 + 12 h 200  6.0  neg.  3.5  neg.
Marlotherm S 5000 + 12 h 200  3.5  neg.  2.5  neg.
Cylophosphamide + 12 h 200  9.0 pos.**  4.0  pos.*
Vehicle control   1 % Acetone  -  20 h  200  3.0  -  2.5  -
 Marlotherm S  1250  -  20 h  200  2.0  neg.  0.5  neg.
 Marlotherm S  2500  -  20 h  200  4.5  neg.  3.0  neg.
 Marlotherm S  5000  -  20 h  200  2.0  neg.  1.5  neg.
 Mitomycin C  0.05  -  20 h  200  14.5  pos.***  10.0  pos.***
Vehicle control   1 % Acetone  +  20 h  200  0.5  -  0.5  -
 Marlotherm S  1250  +  20 h  200  1.0  neg.  0.5  neg.
 Marlotherm S  2500  +  20 h  200  1.5  neg.  1.5  neg.
 Marlotherm S  5000  +  20 h  200  1.5  neg.  0.5  neg.
 Cyclophosphamide  5  +  20 h  200  21.0  pos.***  15.5  pos.***

neg. = statistically negative aberration frequency pos. = statistically positive aberration frequency ** = p<0.01 *** = p<0.001

Tabe #3: Results of Chromosome Aberration assay of preliminary toxicity test slides

    Treatment                Aberrant Cell Frequency   
  Conc. [µg/mL]   S9 Mix  Harvest Time No. of Cells Scored    Including Gaps          Excluding Gaps
           %  Judge  %  Judge
Vehicle Control  1% Acetone  - 12 h 100  0.0  -  0.0  -
Marlotherm S  19.5 - 12 h 100  4.0 neg.  3.0  neg.
Marlotherm S  39 12 h 100  2.0 neg.  0.0  neg.
Marlotherm S 78  12 h 100  0.0 neg.  0.0  neg.
 Marlotherm S 156.25  12 h 100  3.0 neg.  2.0  neg.
Vehicle Control 1% Acetone  + 12 h 100  4.0  -  3.0  -
Marlotherm S 19.5 + 12 h 100  3.0 neg.  3.0  neg.
Marlotherm S 39 + 12 h 100  3.0  neg.  0.0  neg.
Marlotherm S 78 + 12 h 100  9.0  neg.  2.0  neg.
Marlotherm S 156.25  + 12 h 100  3.0 neg.  1.0  neg.
Vehicle control   1 % Acetone  -  20 h  100  3.0  -  0.0  -
 Marlotherm S  19.5  -  20 h  100  4.0  neg.  3.0  neg.
 Marlotherm S  39  -  20 h  100  1.0  neg.  0.0  neg.
 Marlotherm S  78  -  20 h  100  7.0  neg.  3.0  neg.
 Marlotherm S  156.25  -  20 h  100  0.0  neg.  0.0  neg.
Vehicle control   1 % Acetone  +  20 h  100  0.0  -  0.0  -
 Marlotherm S  19.5  +  20 h  100  3.0  neg.  3.0  neg.
 Marlotherm S  39  +  20 h  100  4.0  neg.  1.0  neg.
 Marlotherm S  78  +  20 h  100  4.0  neg.  4.0  neg.
 Marlotherm S  156.25  +  20 h  100  3.0  neg.  1.0  neg.

neg. = statistically negative aberration frequency

Conclusions:
Interpretation of results:
negative

Marlotherm S demonstrated no significant, dose-related increases in the frequency of cells with aberrations either with or without S9 mix. Marlotherm S was shown to be non-clastogenic to CHO cells in vitro.
Executive summary:

The genotoxicity of the test material was evaluated in an in vitro chromosomal aberration study in Chinese Hamster Ovary (CHO) cells in the presence and absence of an external metabolic activation system. Under the conditions of the study the test material was found nto be non-clastogenic.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not mentioned
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
Culture medium: MEM (Gibco) with 10% FCS, 2 mM L-Glutamine, 100 U/100 µ/mL Penicillin/Streptomycin and 1% MEM-NEAA
Incubation medium: culture medium without FCS
- Properly maintained: not mentioned
- Periodically checked for Mycoplasma contamination: yes, tested by DAPI/Hoechst
- Periodically "cleansed" against high spontaneous background: not mentioned
Metabolic activation:
with and without
Metabolic activation system:
microsomal liver enzymes (S9), activation system not mentioned
Test concentrations with justification for top dose:
RANGE-FINDING TEST:
0.019 / 0.038 / 0.075 / 0.15 / 0.3 / 0.6 / 1.25 / 2.5 / 5 mg/mL without S9 mix
0.15 / 0.3 / 0.6 / 1.25 / 1.5 / 5 mg/mL with S9 mix

MAIN TEST:
First main test: 0.15 / 0.3 / 0.6 / 1.25 / 2.5 / 5 mg/ml without S9 mix; 0.3 / 0.6 / 1.25 / 2.5 / 5 mg/ml with S9 mix
Second main test: 0.3 / 0.6 / 1.25 / 2.5 / 5 mg/ml with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: not stated
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
incubation medium containing 1% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation Migrated to IUCLID6: 351 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
incubation medium containing 1% acetone
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
with metabolic activation Migrated to IUCLID6: 20 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Range-finding test: V79 cells were seeded at a density of 1x10E6 cells/flask and incubated at 37°C / 5% CO2 over night. Cells were then incubated for 5 h in the presence of the test article concentrations with and without S9 mix. The test article was diluted in incubation medium (culture medium without fetal calf serum) with 1% acetone. After treatment, cells were washed twice with Hanks, trypsinized and counted. For the cytotoxicity testing cells were adjusted to 10E3 cells/mL, 0.2 mL of this final cell suspension (= 200 cells/dish) were added to 5 mL culture medium in small dishes and incubated for 7 days at 37°C / 5% CO2. Then the cells were fixed with Methanol, stained with 5% Giemsa solution and colonies were counted.
Main test: The treatment of the cells were performed as in the range-finding test. For the expression period treated cells were seeded at a density of 10E6 cells/flask. The survival of the cells after treatment was determined by a cytotoxicity test (see range-finding). The cells were trypsinized after three days and seeded again at 10E6 cells/flask. Six days after treatment the cytotoxicity test was stopped and the number of colonies was determined. The cells in the flasks were harvested and seeded at a density of 0.5x10E6 cells/dish in selection medium. In addition a cytotoxicity test was performed to determine the cloning efficiency. After seven days both assays were terminated and the colonies were counted. The test was repeated in an independent experiment.
- Positive and negative control groups and treatment: Negative and positive controls were treated in parallel with the test material.


DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 6 days (subcultured after 3 days)
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 10 µg/mL 6-Thioguanine

NUMBER OF REPLICATIONS: 1 for the test substance concentrations and positive controls, 3 for the negative controls (2 for the negative control in test 1 without S9)

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

OTHER EXAMINATIONS: none
Evaluation criteria:
The mutant frequency (MF) was calculated from the results of the 6-Thioguanine selection assay. MF is expressed as mutants per 10E6 clonable cells. Mutation frequency values, which were increased in comparison to the negative controls were tested for significance. Only those results, which were significantly different (limit of significance p = 0.05) compared to the highest negative control were considered relevant.
Statistics:
The t-test was used in case of homogenous group variances, otherwise the U-test from Mann and Whitney was used.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
other: yes, except for the first test with S9-mix (because of dilution error of DMBA the vast majority of cells did not survive after treatment)
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined
- Effects of osmolality: not examined
- Precipitation: not occurred
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: In the range finding test with and without S9 mix no significant cytotoxicity of the test article was measured in the range of 0.15 to 5 mg/mL and 0.019 to 5 mg/mL respectively compared to the negative control. Based on these results 5 mg/mL was chosen as highest concentration foth the HPRT test.


COMPARISON WITH HISTORICAL CONTROL DATA: Previous experiments carried out in the testing laboratory indicated a high range of variability within the negative controls (MF-values ranging from 5.7 to 39.1) using the V79 cell culture system.


ADDITIONAL INFORMATION ON CYTOTOXICITY: none
Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

GENOTOXIC EFFECTS:

- With metabolic activation:

First test: The results of all test concentrations analysed were found within the range of the negative controls.

Second test: Treatment with 2.5 and 5.0 mg/mL Marlotherm S resulted in increased MF-values compared to the negative control. The treatment with 5.0 mg/mL Marlotherm S demonstrated a significant difference (p = 0.0032-0.049) compared to all negative controls in the statistical analysis. Since this result was not reproduced in the first test and no dose dependency was measured, it should not be regarded as biologically significant.

- Without metabolic activation:

First test: No increase of the mutation frequency was observed comparing different sample concentrations to the negative controls.

Second test: The concentration of 2.5 mg/mL test substance resulted in an increase of mutant cells compared to the range of the negative controls. The statistical analysis of the MF-value in the U-test revealed no significant difference compared to the highest negative control.

In all tests performed, the positive controls induced a significant response under the chosen conditions except for the first test with S9-mix. Because of dilution error of DMBA the vast majority of cells did not survive after treatment (CE of 0 at expression period).

Table #1: Range-finding test

concentration [mg/mL]   S9 mix  Mean colony number ± SD  Cloning efficiency [%]
 Neg. Control  -  144 ± 13  72
 0.019  -  118 ± 10  59
 0.038  -  104 ± 5  52
 0.075  -  127 ± 21  64
 0.15  -  141 ± 4  71
 0.3  -  109 ± 16  55
 0.6  -  157 ± 12  79
 1.25  -  139 ± 10  70
 2.5  -  121 ± 5  61
 5 -  127 ± 16  64
 Neg. Control  + 105 ± 15  53
 0.15  +  93 ± 20  47
 0.3  +  122 ± 20  61
 0.6  +  112 ± 13  56
 1.25  +  118 ± 17  59
 2.5  +  122 ± 6  61
 5  +  134 ± 9  67

Table #2: HPRT Test

concentration [mg/ml] MF [mutants/10E6 clonable cells]          
  test #1 -S9   test #1 +S9  test #2 -S9  test #2 +S9
 Neg. control (1)  7.3  9.5  14.1  10.4
 Neg. control (2)  35.4  18.8  21.6  5.7
 Neg. control (3) n.p.   15.6  12.8  17.8
 0.15  12.9  n.p.   n.p.   n.p.
 0.3  6.1  12.6  18.9  15.1
 0.6  9.4  18.2  17.2  15.8
 1.25  16.7  9.0  16.9  12.7
 2.5  3.7  9.8  38.1  21.8
 5  15.4  18.4  20.5 24.7*
Pos. control (EMS)  166.2   n.p.  192.7   n.p.
 Pos. control (DMBA) n.p.  0.0**   n.p.  220.1

n.p.= not performed

* statistically significant

** dilution error of positive control, cells did not survive after treatment

Conclusions:
Interpretation of results:
negative

In conclusion, the results of all experiments indicated that the test article Marlotherm S had no reproducible biologically significant effect on the mutation frequency in the HPRT-locus under the test conditions described in this study report.
Executive summary:

The test material was tested in an in vitro gene mutation test in Chinese hamster lung fibroblasts (V79) in the presence and absence of a metabolic activation system. Under the conditions of the study the test material was found to be not mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this guideline.
Deviations:
yes
Remarks:
Individual body weights at study start were not indicated; some parameters, as age of animals at study start and humidity level during study, were not reported; sampling time was only 6 hours following end of treatment.
GLP compliance:
no
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: COBS CD-1 (ICR) BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: no data
- Weight at study initiation: 18-21 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: 2 (preliminary study) or 5 (main study) animals/sex/cage
- Diet (e.g. ad libitum): laboratory diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): no ndata
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12


IN-LIFE DATES: no data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: UGILEC 102 was miscible with corn oil
- Concentration of test material in vehicle: 100, 200 and 400 mg/mL
- Amount of vehicle (if gavage or dermal): animals in all groups were dosed with the standard volume of 0.1 ml/10g bodyweight (10 mL/kg bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ugilec 102 was prepared as a solution in corn oil using a high speed mixer.
Duration of treatment / exposure:
24h.
Frequency of treatment:
2 administrations separated by an interval of 24h.
Post exposure period:
6 hours
Remarks:
Doses / Concentrations:
1000, 2000 and 4000 mg/kg/day for 2 days
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s): yes, a known mutagen (recommended by OECD for this specific test)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg
Tissues and cell types examined:
Tissues: bone marrow smear from each femur
Cells: erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
based on the Maximal Tolerated Dose obtained from preliminary toxicity testing.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling was performed on dead animals sacrificed 6 hours after the last administration.

DETAILS OF SLIDE PREPARATION:
Following animal sacrifice, a direct bone marrow smear was made onto a slide containing a drop of calf serum. The slide was cleaned by immersion in methanol for 24 hours and air-dried immediately before use. One smear was made from each femur. The prepared smears were air-dried and fixed in methanol overnight. After fixation, the smears were air-dried and placed in buffered distilled water (pH 6.8) for 10 minutes prior to staining in Giemsa (dilution factor; 1 part Giemsa : 9 parts buffered distilled water, pH 6.8) for 10 minutes. After rinsing in buffered distilled water (pH 6.8), the slides were air-dried and mounted in DPX.

METHOD OF ANALYSIS:
The stained smears were coded and examined by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal and the ratio of normochromatic to polychromatic erythrocytes.
Evaluation criteria:
No data
Statistics:
No data
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
from a total dose of 4000 mg/kg
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Phase I (2 animals/sex/dose): 2 administrations from 250 to 10400 mg/kg bw/d (from 500 to 20800 mg/kg bw in total). Phase II (5 animals/sex/dose): 2 administrations from 4500 to 6500 mg/kg bw/d (from 9000 to 13000 mg/kg bw in total)
- Mortality: (see Table 1 in the field Remarks on results)
- Clinical signs of toxicity: After administration of corn oil, the vehicle control, no toxic reactions were observed. After administration of Ugilec 102 at a total dosage of 500 mg/kg bodyweight, no toxic reactions were observed. At a total dosage of 5000 mg/kg bodyweight, no toxic reactions were observed after the first dose until the morning of the second day (24 hours after the first dose) when hunched posture, lethargy and pale external extremities were observed. In addition, all animals in this group were observed to be thinner than those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose. At total dosages between 9000 and 20800 mg/kg bodyweight, a toxic reaction consisting of lethargy was observed 1 hour after the first dose and was still present 7 hours later. On the morning of the second day (24 hours after the first dose), the lethargy was still present together with hunched posture, pale external extremities and ptosis. All animals in each group were observed to be thinner thon those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose.



RESULTS OF DEFINITIVE STUDY (see Table 2 in the field Remarks on results)
- Mortality: no mortality was observed in the low dose animal group. Two females treated at 2000 mg/kg bw/d (4000 mg/kg bw in total) were found dead 6 hours after the second treatment. Macroscopic examination at post mortem of these two unexpected deaths, failed to reveal any abnormalities. Four animals (two females and two males) treated at 4000 mg/kg bw (8000 mg/kg bw in total) were found dead 5 or 6 hours respectively, after the second dose. Macroscopic examination at post mortem of all four animals, failed to reveal any abnormalities.
- Clinical signs of toxicity: After administration of corn oil, the vehicle control, no toxic reactions or deaths were observed.
After administration of Ugilec 102 at a total dosage of 2000 mg/kg bw/d, no toxic reactions were observed. At a total dosage of 4000 mg/kg bodyweight, no toxic reactions were observed after the first dose until the morning of the second day (24 hours after the first dose) when hunched posture, lethargy and pale external extremities were observed. In addition, aIl animals in this group were observed to be thinner thon those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose. At a total dosage of 8000 mg/kg bw/d, a toxic reaction consisting of lethargy was observed 1 hour after the first dose and was still observed 7 hours later. On the morning of the second day (24 hours after the first dose) the lethargy was still present together with hunched posture, pale external extremities and ptosis. All animals in this group were observed to be thinner than those in the control group. The symptoms were still present at the time of sacrifice, 6 hours after the second dose.

- Induction of micronuclei (for Micronucleus assay): after administration of Ugilec 102 at all total dosages, the group mean micronucleated cell counts were comparable with the concurrent control value.
- Ratio of NCE/PCE (for Micronucleus assay): After administration of Ugilec 102 at all total dosages, the group mean normochromatic to polychromatic cell ratios were comparable with the concurrent control value.
- Appropriateness of dose levels and route: toxic reactions observed in the micronucleus test as expected as they were similar to the reactions observed in preliminary toxicity study at the same dose levels.
Conclusions:
Interpretation of results: negative
UGILEC 102 (Dibenzylbenzene, ar-methyl derivative) did not present any in vivo genotoxic activity in the micronucleus test conducted in mice orally gavaged twice at 1000, 2000 and 4000 mg/kg bw.
Executive summary:

The genotoxicity of the test substance, Dibenzylbenzene, ar-methyl derivative (UGILEC 102) was determined in a micronucleus test conducted in vivo with COBS CD-1 (ICR) BR mice using a similar method to the OECD Guideline 474.

Solutions were administered by gavage at a constant volume of 10 mL/kg bw. Following preliminary toxicity assay, 4000 mg/kg bw/d treatment for 2 days was selected as the maximum tolerated dose, based on high rates of mortality and severe clinical signs observed at higher dosages.

Doses of 1000, 2000, and 4000 mg/kg bw/d (2000, 4000 and 8000 mg/kg bw in total) were administered by oral gavage to groups of 10 mice (5 males and 5 females), in two equal dosages, separated by an interval of 24 hours. Mitomycin C, administered intraperitoneally at a total dose level of 8 mg/kg, served as positive control.

Animal were sacrificed 6 hours after the second administration, and bone marrow smears examined for the presence of micronuclei in 2000 polychromatic erythrocytes per mouse and for the ratio NCE/PCE. No significant increased mortality was observed in any treated groups.

Signs of toxicity were only observed at the dose of 2000 mg/kg bw/d (4000 mg/kg bw in total, respectively), consisting of hunched posture, lethargy, pale external extremities and also ptosis at the dose-level of 8000 mg/kg bw in total. The NCE/PCE ratio obtained in all treated animals was similar to that of control animals. The number of micronucleated polychromatic cells in animals given dibenzyltoluene was similar to that obtained in control animals. Conversely, animals treated with Mitomycin C presented a highly significant increase in the number of micronucleated polychromatic cells. The test substance was concluded to be negative in the micronucleus test with mice.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From December 13, 1989 to April 20, 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Comparable to guideline study with acceptable restrictions: - 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:
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
Specific details on test material used for the study:
- Name of the test material (as cited in study report): JARYLEC DBT
- Stability under test conditions: found to be stable in dosing solutions (suspensions in 10% aqueous gum arabic solutions) by analysis of content in solutions at approximately 3 months, 4 months, and 4 months and 2 weeks after the start of the treatment.
- Batch number: 5807
- Composition: Dibenzyltoluenes (97%), tribenzyltoluenes (1.9%), benzyltoluene (< 0.05%).
- Impurities: dibenzylxylenes (0.9%), dibenzylbenzenes (0.2%), anthracene (95 ppm), benzene (35 ppm), toluene (35 ppm), xylenes (< 10 ppm).
Species:
rat
Strain:
Sprague-Dawley
Sex:
male
Details on test animals 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
Remarks:
Doses / Concentrations:
500 mg/kg/day for 4 months
Basis:
nominal conc.
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:
Interpretation of results: negative
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:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
not mentioned
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Specific details on test material used for the study:
The purity of the material used for the test was lower than the purity of the actual material. It contained ca. 26% of isomers of dimethyl tribenzenes. However, based on comparable basic structure of the impurities and the assumption that any toxic effects would increase with higher molecular weight and lipophilicity, we regard that the studies conducted with the lower purity material being "worst case" and can be used for the evaluation of the actual product.
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann Versuchstierzucht, Borchen, Germany
- Age at study initiation: adult (about 3 month)
- Weight at study initiation: 31.0 ¿ 40.4 g (males); 28.9 ¿ 34.4 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: not mentioned
- Housing: collective housing in Macrolon cages type II, max. 5 animals per cage
- Diet (e.g. ad libitum): Ssniff-R complete diet ad libitum, supplied by Ssniff Spezialdiäten GmbH, Soest, Germany
- Water (e.g. ad libitum): drinking water as for human consumption ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: not mentioned
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (Roth GmbH & Co. KG, Karlsruhe, Germany)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: The test solution was prepared by dissolving an appropriate amount in vehicle, no further details mentioned
Frequency of treatment:
single intraperitoneal injection
Post exposure period:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:
6000 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): not stated
- Route of administration: single intraperitoneal injection
- Doses / concentrations: 40 mg/kg b.w., application volume 10 mL/kg b.w.
Tissues and cell types examined:
Preparations of the bone marrow were made on slides. Examination of poly- and normochromatic erythrocytes and micronucleated cells.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
On the basis of theresults of the range finding study, the dose of 6000 mg/kg body weight was considered to be near the maximal tolerated dose (MTD) and was therefore chosen for the main study.


DETAILS OF SLIDE PREPARATION:
The animals were killed by cervical dislocation 24, 48 or 72 hours after treatment. The femora were removed and the bone marrow was suspended in fetal calf serum. Samples were centrifuged at 1600xg and subsequently decanted. One drop of each suspension was then smeared on a slide by means of a second slide. Two preparations were made from each animal, dried, fixed in absolute (99%) methanol for 5 min and then allowed to dry in air. Slides were stained with a May-Gruenwald and Giemsa solution. Prior to analysis all slides were randomized and coded (blind evaluation).


METHOD OF ANALYSIS:
The cells were examined under a microscope at a thousandfold magnification. A total of 1000 polychromatic erythrocytes were examined on each slide and the number of micronucleated cells in each sample were recorded. The ratio of polychromatic to normochromatic (mature) erythrocytes was calculated for a sample of 1000 cells.

OTHER:
Clinical observations of the treated animals were made for the justification for dose selection.
Evaluation criteria:
Cell counts were based on a total of 1000 cells per animal. Historical control data from the labotary indicated that an incidence of up to 8 micronucleated cells per 1000 polychromatic erythrocytes may be considered to be within normal limits.
Statistics:
t-tests for independent samples in each test group and the corresponding negative control group with the number of polychromatic erythrocytes with micronuclei, number of polychromatic erythrocytes, number of normochromatic erythrocytes, ratio of poly- to normochromatic erythrocytes.
Comparison of mean values of the negative and positive control groups with the Mann-Whitney U-test.
Significance levels were indicated as follows: * p < 0.05; ** p < 0.01; *** p 0.001
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY

- Dose range: 2500, 5000, 7500 and 10 000 mg/kg bw, repetition of range finding with 6000 mg/kg bw ; single intraperitoneal injections to each 2 male and 2 females.
- Clinical signs of toxicity in test animals: In the highest-dosed group (10,000 mg/kg bw), all mice (2 male, 2 female) were found dead 72 hours p.a. 2 male mice treated with 7500 mg/kg died within the 72 h observation period. Frequent clinical findings in animals treated with 10,000 and 7500 mg/kg were reduced activity, nociceptive responsiveness, abdominal tone, skin turgor, pinna-reflex and respiratory rate; abnormal gait, prone position, cyanosis and piloerection were also frequently observed. Animals receiving 5000 and 2500 mg/kg revealed no abnormal clinical signs. In the repetion of the range finding with 6000 mg/kg slight to moderate piloerection and a slight decrease in activity were the only clinical signs observed.
- Evidence of cytotoxicity in tissue analyzed: not examined


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: The number of polychromatic erythrocytes with micronuclei was significantly increased 24h post-injection in the positive control animals. In relation to the untreated controls, the number of polychromatic erythrocytes with micronuclei in the test groups did not differ significantly.
- Ratio of PCE/NCE: The ratio of poly- to normochromatic erythrocytes in one test group (6000 mg/kg b.w., males, 72 h) differed significantly to the corresponding control animals, on the basis of historical control data, this result was within the normal range.
- Appropriateness of dose levels and route: The ratio of poly- to normochromatic erythrocytes in the test group showed that the test substance reached the bone marrow and caused a slight toxic effect. Frequent clinical findings in animals treated with a single dose of 6000 mg/kg b.w . were piloerection and depressed activity. An irregular respiratory rate was observed occasionally in some animals. In most animals the flanks appeared hollow over the entire observation period. No animal died in the main study.
- Statistical evaluation: In group III males (72 h, 6000 mg/kg bw) the PCE/NCE ratio was significantly decreased relative to the corresponding control animals (p < 0.01; t-test for independent samples).
In the positive control animals the PCE number was significantly increased (p < 0.01; U-test), demonstrating the sensitivity of the chosen test system.

Table #1: Results of in vivo micronucleus test for male animals

      Neg. control   test substance 6000 mg/kg       Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
 number of cells evaluated     1000  1000  1000  1000  1000  1000  1000
 number of erythrocytes  normochromatic  569.6 ± 70.2  605.4 ± 78.5  601.8 ± 51.2  632.8 ± 123.8  653.8 ± 97.7  738.4 ± 43.9**  512.0 ± 42.4
   polychromatic 430.4 ± 70.2  394.6 ± 78.5  398.2 ± 51.2  367.2 ± 123.8  346.2 ± 97.7  261.6 ± 43.9**  488.0 ± 42.4
   polychromatic with micronuclei  1.0 ± 1.0  1.4 ± 1.1 1.2 ± 1.3 1.2 ± 0.8   0.6 ± 0.9  1.0 ± 0.7  38.2 ± 12.0**
 ratio of erythrocytes  polychromatic /normochromatic 0.776 ± 0.205  0.674 ± 0.227  0.672 ± 0.144  0.622 ± 0.280  0.554 ± 0.217  0.358 ± 0.080**  0.964 ± 0.166

** = statistically significant (p < 0.01 U-test (Mann-Whitney))

Table #2: Results of in vivo micronucleus test for female animals 

      Neg. control   test substance 6000 mg/kg       Pos. control
  sampling time    24 h  48 h  72 h  24 h  48 h  72 h 24 h 
 number of cells evaluated     1000  1000  1000  1000  1000  1000  1000
 number of erythrocytes  normochromatic 573.8 ± 94.5  538.0 ± 83.6  529.4 ± 82.1  545.8 ± 30.8 572.0 ± 59.6  535.4 ± 69.1  510.8 ± 50.5
   polychromatic 426.2 ± 94.5  462.0 ± 83.6  470.6 ± 82.1  454.2 ± 30.8  528.0 ± 59.6  464.6 ± 69.1  489.2 ± 50.2
   polychromatic with micronuclei  1.4 ± 1.7  0.8 ± 1.3  1.2 ± 1.3  0.6 ± 0.5  1.2 ± 1.6  0.4 ± 0.5  25.4 ± 4.7**
ratio of erythrocytes  polychromatic/normochromatic  0.782 ± 0.304  0.898 ± 0.318  0.932 ± 0.333  0.836 ± 0.099  0.764 ± 0.178  0.894 ± 0.264  0 .976 ± 0.215

** = statistically significant (p < 0.01 U-test (Mann-Whitney))

Conclusions:
Interpretation of results: negative
A single intraperitoneal administration of the test substance at a dose of 6000 mg/kg bw to male and female mice did not produce a significant increase in the frequency of micronuclei in the polychromatic erythrocyte cells.The mean of all parameters were within the respective normal range of the historical data of the testing laboratory. Therefore, under the experimental conditions of this study the test substance is considered to be non-mutagenic.
Executive summary:

An in vivo micronucleus assay was conducted with the test material. Under the conditions of the study the test substance was considered to be not mutagenic.

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.


Short description of key information:
The mutagenicity of Dibenzylbenzene, ar-methyl derivative has been evaluated in various studies in vitro and in vivo. The available information clearly indicate that the material is not gentoxic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

No classification for mutagenic effects is indicated according to the general classification and labeling requirements for dangerous substances and preparations (67/544 EEC) or the classification, labeling and packaging (CLP) regulation (EC 1272/2008).