Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro genotoxicity in bacteria

 

In a well conducted study (Schöberl, 1994) (performed according to EU Method B.13/14 and GLP compliant), the genotoxicity of benzyl toluene was determined in a reverse mutation assay conducted in vitro with salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 at the doses of 8, 40, 200, 1000 and 5000 µg/plate with and without a metabolic activation system prepared from a liver microsomal fraction of rats. The test substance did not induce any significant increase in the number of revertants, with or without S9 mix, in any of the 5 tested strains.

 

These results are supported by those of two another well conducted study (Richold et al., 1981; Scholz, 1990).

The potential ability of benzyl toluene to induce point mutations in bacteria was investigated in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without a metabolic activation system prepared from a liver microsomal fraction of rats. In the Richold’s study, benzyl toluene was tested in triplicate, by direct plate incorporation, at the concentrations of 1.25 to 10 µL/plate for strain TA1535 and 0.0025 to 0.02 µL/plate for strains TA1537, TA1538, TA98 and TA100. In the Scholz’ study benzyl toluene was tested at concentrations of 8, 40, 200, 1000 and 5000 µg/plate by plate incorporation test and 125, 250, 500, 1000 and 2000 µg/plate by the preincubation method. The number of revertants induced by the positive controls were significantly higher than the spontaneous one, which demonstrated the sensitivity of the test strains and the efficiency of the metabolic activation system. No significant increase in the revertant number was observed whatever the strain tested. Under these experimental conditions, benzyl toluene was not genotoxic in the Ames test.

 

In all of those studies, although E. coli WP2 or S. typhimurium TA102 were not tested and although conditions of testing were not always sufficiently described -especially for supporting studies- (test medium, incubation time and temperature were not described), tests were considered as valid, considering there were conducted in accordance with standard guidelines, applicable at the time when test was conducted (1981).

 

In vitro genotoxicity in mammalian cell

Benzyl toluene was tested for in vitro mammalian gene mutation test in V79 cells in a GLP OECD 476 study (Creutziger, 1990). With metabolic activation, both experiments (concentrations of 0.075 and 0.150 mg/mL) showed increased mutation frequencies. Only in the first test the result of the higher concentration (0.150 mg/mL) was statistically significant (p = 0.001 to 0.0018) in the U-test. This result however was not judged to be biologically relevant, since this MF value was within the range of historical control data of the testing laboratory (MF-values ranging from 5.7 to 39.1). In conclusion the results of all experiments indicated that the benzyl toluene had no reproducible biologically significant effect in the mutation frequency in the HPRT-locus with and without S-9.

Link to relevant study records

Referenceopen allclose all

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:
1994-10-20 to 1994-12-27
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, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
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 and 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, TA 1538; Sodium azide (2.5 µg/plate) for TA 100, TA 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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and 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
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: yes, at concentrations 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:
- With metabolic activation:
Plate incorporation test: background lawn reduced at 200 µg/plate and complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1535); background lawn reduced at 1000 µg/plate and complete clearing of background lawn at 5000 µg/plate (TA 1537)
Incubation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1537)
- Without metabolic activation:
Plate incorporation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); background lawn reduced at 200 µg/plate and complete clearing of background lawn at 1000 and 5000 µg/plate (TA 1537)
Incubation test: background lawn reduced at 200, 1000 and 5000 µg/plate (TA 1535); complete clearing of background lawn at 200, 1000 and 5000 µg/plate (TA 1537)

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*

 35

 54

 no

 132

 143

 no

 16

 18

 no

8

 33

 62

 no

 140

 156

 no

 20

 16

 no

40

 33

 46

 no

 141

 144

 no

 17

 17

 no

200

 31

 50

 no

 144

 140

 no

 13

 12

 yes

1000

 38

 46

 no

 115

 136

 no

 11

 8

 yes

5000

 25

 50

 no

 97

 113

 no

 9

 3

 yes

Positive control

 156

 2153

 no

 405

 1994

 no

 445

 236

 no

*solvent control with DMSO

Table #2: 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*

 15

 17

 no

 27

 34

 no

 

 

 

8

 18

 14

 no

 28

 32

 no

 

 

 

40

 12

 11

 no

 23

 36

 no

 

 

 

200

 2

 11

 yes

 26

 39

 no

 

 

 

1000

 0

 5

 yes

 24

 49

 no

 

 

 

5000

 0

 0

 yes

 25

 32

 no

 

 

 

Positive control

 901

 264

 no

 178

 1428

 no

 

 

 

*solvent control with DMSO  

Table #3: 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*

 30

 40

 no

 150

 147

 no

 10

 15

 no

8

 31

 45

 no

 151

 166

 no

 11

 15

 no

40

 40

 49

 no

 145

 146

 no

 8

 15

 no

200

 33

 45

 no

 147

 159

 no

 5

 16

 yes

1000

 32

 43

 no

 120

 138

 no

 1

 6

 yes

5000

 31

 47

 no

 100

 105

 no

 2

 2

 yes

Positive control

 162

 1669

 no

 432

 2081

 no

 354

 199

 no

*solvent control with DMSO 

Table #4: 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*

 22

 20

 no

 21

 28

 no

 

 

 

8

 13

 21

 no

 24

 38

 no

 

 

 

40

 15

 20

 no

 19

 43

 no

 

 

 

200

 0

 22

 yes

 24

 31

 no

 

 

 

1000

 0

 5

 yes

 23

 34

 no

 

 

 

5000

 0

 0

 yes

 18

 32

 no

 

 

 

Positive control

 68

 272

 no

 169

 864

 no

 

 

 

*solvent control with DMSO 

Conclusions:
Benzyltoluene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella.
Executive summary:

The potential of benzyltoluene to induce reverse mutation in Salmonella typhimurium (strains: TA 1535, TA 1537, TA 98, TA 100) was evaluated in accordance with the international guidelines (OECD 471, ) and in compliance with the Principles of Good Laboratory Practice. Benzyltoluene was tested, with and without a metabolic activation system; bacterias were exposed to benzyltoluene at 8, 40, 200, 1000 and 5000 µg/plate. Nonoteworthy increase in the number of revertantswas observed for all doses with and without metabolic activation on the 5 tested strains. Under our experimental conditions, benzyltoluene did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella. typhimurium.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting 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 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Study conducted prior to the adoption of the most recent version of this Guideline
Deviations:
yes
Remarks:
E. coli WP2 or S. typhimurium TA102 was not tested; conditions of testing are not sufficiently described (test medium, incubation time and temperature were not described); Only 4 concentrations instead of at least 5 were tested.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
This test enables the detection of base-pair substitution and frameshift mutagens. Mutagenic substances can induce reversion in histidine-deficient strains, which are then able to grow and form colonies in a histidine-limited medium, while non-revertants can not.
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
TA 1535: 0, 1.25, 2.5, 5 and 10 µL/plate.
Others strains: 0, 0.0025, 0.005, 0.01 and 0.02 µL/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: no
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Remarks:
With or without S9
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Used for TA 1535 and TA 100 Without S9
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine
Remarks:
Used for TA 1537, TA 1538, TA 98 Without S9
Positive controls:
yes
Positive control substance:
other: 2-amino-anthracene
Remarks:
Used for TA 1535, TA 98 and TA 100 With S9
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Used for TA 1538 With S9
Positive controls:
yes
Positive control substance:
other: neutral red
Remarks:
Used for TA 1537 With S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION not reported


SELECTION AGENT (mutation assays): Histidine


NUMBER OF REPLICATIONS: 3 plates/concentration


DETERMINATION OF CYTOTOXICITY
- other: observation whether the bacterial lawn is sparse and whether the number of colonies is decreased.


OTHER
The method used is described in HRC Protocol MCB 101.
Evaluation criteria:
No data
Statistics:
No data
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537 & TA1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Remarks:
only reported for preliminary dose range-finding testing
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS: no data


RANGE-FINDING/SCREENING STUDIES:
0.1, 1 and 10 µl/plate in all strains; no toxicity towards TA1535 up to 10 µL/plate


COMPARISON WITH HISTORICAL CONTROL DATA: no data

Table 1: Mean Revertants per Plate

Test

Material

TA-1535

TA-1537

TA-1538

TA-98

TA-100

Mean

Mean

Mean

Mean

Mean

Nonactivation

Solvent Control

16

11

13

21

96

0.0025 µL/plate

-

9

14

27

82

0.005 µL/plate

-

9

7

17

66

0.01 µL/plate

-

5

6

15

65

0.02 µL/plate

-

9

11

19

IL

1.25 µL/plate

17

-

-

-

-

2.5 µL/plate

12

-

-

-

-

5 µL/plate

10

-

-

-

-

10 µL/plate

3

-

-

-

-

Activation

Solvent Control

14

19

13

20

88

0.0025 µL/plate

-

18

14

23

93

0.005 µL/plate

-

19

7

25

87

0.01 µL/plate

-

14

6

19

77

0.02 µL/plate

-

15

11

18

63

1.25 µL/plate

14

-

-

-

-

2.5 µL/plate

15

-

-

-

-

5 µL/plate

7

-

-

-

-

10 µL/plate

7

-

-

-

-

IL: Incomplete bacterial lawn

-: test not conducted

Conclusions:
The test substance was found to be negative in the in vitro genotoxicity Ames assay both in the presence or absence of metabolic activation.
Executive summary:

The potential ability of benzyl toluenes to induce point mutations in bacteria was investigated by the Ames test in five strains of Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100, with and without a metabolic activation system prepared from a liver microsomal fraction of rats. The number of revertants induced by the positive controls were significantly higher than the spontaneous one, which demonstrated the sensitivity of the test strains and the efficiency of the metabolic activation system. Benzyl toluenes was tested in triplicate, at the concentrations of 1.25 to 10 µg/plate for strain TA1535 and 0.0025 to 0.02 µg/plate for strains TA1537, TA1538, TA98 and TA100. No significant increase in the revertant number was observed whatever the strain tested. Under these experimental conditions, benzyl toluenes was not genotoxic in the Ames test.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1990-02-21 until 1990-04-09
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)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced liver S9 mix of male Wistar rats
Test concentrations with justification for top dose:
8, 40, 200, 1000 and 5000 µg/plate plate incorporation test
125, 250, 500, 1000 and 2000 µg/plate preincubation test
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 (50 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:
other: only tested with strain TA 100
Positive control substance:
other: Aminoanthracene (10 µg/plate) with strainTA 100
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
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
TA 1535 >= 200 µg/plate; TA 1537 >= 1000 µg/plate
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: 125 µg/plate and higher concentrations in the preincubation test, 200 µg/plate and higher in the plate incorporation test
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: not performed


COMPARISON WITH HISTORICAL CONTROL DATA: not performed


ADDITIONAL INFORMATION ON CYTOTOXICITY: none

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*

 31±4

 57±4

 no

 279±24

 169±6

 no

 16±6

 18±4

 no

8

 40±1

66±11 

 no

 282±7

 199±3

 no

 18±4

 16±4

 no

40

 36±8

 66±12

 no

 288±18

 156±9

 no

 12±8

 11±6

 no

200

 41±5**

 61±4**

 no

 287±19**

 179±35**

 no

 11±2**

 0±0**

no (- MA) 

yes (+ MA)

1000

 38±11**

 36±5**

 no

 248±3**

 130±11**

 no

 6±4**

 0±0**

no (- MA) 

yes (+ MA)

5000

 27±8**

 41±5**

 no

 305±33**

 137±12**

 no

 5±2**

 0±0**

 yes

Positive control

 414±17

 not tested

 no

 678±67

 1667±667

 no

 370±9

 not tested

 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*

 11±4

 13±7

 no

 43±2

 60±9

 no

 

 

 

8

 8±2

 21±9

 no

 41±8

 69±17

 no

 

 

 

40

 5±3

 23±3

 no

 34±5

 60±4

 no

 

 

 

200

 6±4**

 10±4**

 no

 41±5**

 56±5**

 no

 

 

 

1000

 0±0**

 0±0**

 yes

 47±6**

 65±10**

 no

 

 

 

5000

 0±0**

 0±0**

 yes

 46±8**

 61±8**

no

 

 

 

Positive control

 531±218

 not tested

 no

 257±83

 not tested

 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*

 44±9

 76±8

 no

 302±19

 176±13

 no

 18±6

 21±9

 no

125

 46±3**

 56±5**

 no

 300±22**

 198±5**

 no

 13±3**

 19±5**

 no

250

 48±7**

 68±2**

 no

 256±61**

 189±5**

 no

 15±7**

 17±10**

 no (- MA) yes (+ MA)

500

 45±6**

 68±11**

 no

 287±24**

 181±21**

 no

 13±7**

 14±7**

 no (- MA) yes (+ MA)

1000

 52±5**

 78±8**

 no

 247±25**

 162±6**

 no

 23±5**

 13±2**

 no (- MA) yes (+ MA)

2000

 44±5**

 68±6**

 no

 273±19**

 146±11**

 no

 19±4**

 10±5**

 no (- MA) yes (+ MA)

Positive control

 280±13

 not tested

 no

 747±50

 2795±101

 no

 389±51

 not tested

 no

*solvent control with DMSO ** precipitation  

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*

 20±3

 21±4

 no

 43±8

 57±6

 no

 

 

 

125

 18±2**

 28±7**

 no

 48±11**

 57±6**

 no

 

 

 

250

 16±3**

 32±2**

 no

 45±3**

 67±11**

 no

 

 

 

500

 14±3**

 18±4**

 no

 50±5**

 73±18**

 no

 

 

 

1000

 16±5**

 14±6**

 yes

 50±11**

 67±5**

 no

 

 

 

2000

 16±3**

 10±4**

 yes

 51±11**

 59±1**

 no

 

 

 

Positive control

 499±176

 not tested

 no

 141±10

 not tested

 no

 

 

 

*solvent control with DMSO ** precipitation  

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
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-Glutamin, 100 U/100 µg/mL Penicillin/Streptomycin and 1 % MEM-NEAA
Incubation medium: culture medium without FCS
- Properly maintained: not mentioned
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not mentioned
- 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
0.005 / 0.010 / 0.019 / 0.038 / 0.075 / 0.15 mg/mL with S9 mix
additional range-finding with S9 mix: 0.018 / 0.038 / 0.075 / 0.15 / 0.3 / 0.6 / 1.25 / 2.5 and 5 mg/ml

MAIN TEST:
0.010 / 0.019 / 0.038 / 0.075 / 0.15 mg/ml with and without S9 mix (and 0.005 mg/ml only in the first test without S9)
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:
concentration in the test: 351 µg/mL Migrated to IUCLID6: without S9 mix
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:
concentration in the test: 20 µg/mL Migrated to IUCLID6: with S9 mix
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 medium containing 10 µg/mL 6-Thioguanine as selective agent. In addition a cytotoxicity test was performed to determine the cloning efficiency. After seven days both assays were terminated and the colonies were counted.
- Positive and negative control groups and treatment: Negative and positive controls were treated in parallel with the test material.
Negative control: incubation medium with 1% acetone.

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:
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.
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: The test substance induced cytotoxic effects in the presence of metabolic activation (S9 mix) in doses of 5, 2.5 and 1.25 mg/mL. At concentrations of 0.6 and 0.3 mg/mL the cloning efficiency was significantly reduced.
In the range finding test without metabolic activation a significant toxicity was measured in the range of 0.3 to 5 mg/mL compared to the negative control.

COMPARISON WITH HISTORICAL CONTROL DATA: see below

GENOTOXIC EFFECTS:
- With metabolic activation: In both experiments concentrations of 0.075 and 0.150 mg/mL showed increased mutation frequencies. Only in the first test the result of the higher concentration (0.150 mg/mL) was statistically significant (p = 0.001 to 0.0018) in the U-test. This result however was not judged to be biologically relevant, since this MF value was within the range of historical control data of the testing laboratory (MF-values ranging from 5.7 to 39.1).
- Without metabolic activation:
First test: No increase of the mutation frequency was observed comparing different sample concentrations to the negative controls.
Second test: Concentrations of 0.010 and 0.019 mg/mL test substance resulted in an increase of mutant cells. The statistical analysis of the MF-values in the U-test revealed no significant difference compared to the highest negative control of either of both results.
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: Mutation frequencies of test substance and controls

concentration [mg/mL]  mutants/10E6 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.   9.5  12.8  17.8
 0.005  15.7  n.p.   n.p.   n.p.
 0.010  10.8  12.4  27.3  17.4
 0.019  4.0  11.9  27.3  17.1
 0.038  8.6  11.8  10.1  12.1
 0.075  8.6  24.5  20.0  26.4
 0.150  20.2  30.8*  17.3 22.0
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:
The test article had no repoducible biologically significant effect in the mutation frequency in the HPRT-locus with and without S-9 under the test conditions described in the study report.
Executive summary:

Benzyltoluene was tested for in vitro mammalian gene mutation test in a GLP OECD 476 study. With metabolic activation, both experiments (concentrations of 0.075 and 0.150 mg/mL) showed increased mutation frequencies. Only in the first test the result of the higher concentration (0.150 mg/mL) was statistically significant (p = 0.001 to 0.0018) in the U-test. This result however was not judged to be biologically relevant, since this MF value was within the range of historical control data of the testing laboratory (MF-values ranging from 5.7 to 39.1). In conclusion the results of all experiments indicated that the test article had no repoducible biologically significant effect in the mutation frequency in the HPRT-locus with and without S-9 under the test conditions described in the study report.

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

Genetic toxicity in vivo

Description of key information

Three micronucleus assays are available.

 

In a key study, the genotoxicity of benzyl toluene (Ugilec 101) 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 (Richold and Richardson, 1981). Solutions were administered by gavage at a constant volume of 10 mL/kg bw. Following preliminary toxicity assay, 500 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 125, 250, and 500 mg/kg bw/d 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 500 mg/kg bw/d, consisting of hypopnea, ptosis, ataxia and tremors. 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 benzyl toluene 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. Benzyl toluene was concluded to be negative in the micronucleus test with mice.

 

As part of the 4-month toxicity study (Verschuere, 1992) of benzyl toluene (JARYLEC BT in the report) administered orally (0, 5, 50, and 500 mg/kg/day) to Sprague-Dawley rats, it was determined whether this compound induced micronuclei in bone marrow erythrocytes (Verschuere, 1991). 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 group treated at 500 mg/kg/day. No increase in the number of micronucleated, normochromatic or polychromatic cells, and no significant decrease in the PCE/NCE ration were noted. Benzyl toluene did not present any in vivo genotoxic activity in the micronucleus test after a 4-month treatment in the rat at 500 mg/kg/day.

 

Benzyl toluene was tested in a mammalian micronucelus test (OECD 474 study, GLP) (Creutziger, 1990). A single intraperitoneal administration of the test substance at a dose of 600 mg/kg bw to male and female mice produced a slightly significant increase in the frequency of micronuclei in polychromatic erythrocyte cell 24h after administration. According to the historical data of the testing laboratory the mean value of this parameters measured were within the normal range. Under the experimental conditions of this study, the test substance was considered to be non-mutagenic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
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 bodyweights 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 arrival: 18 - 21 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, overnight
- Housing: 2 or 5 animals/sex/cage
- Diet: laboratory diet ad libitum
- Water: tap water ad libitum
- Acclimation period: 10 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): no data
- 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 101 was miscible with corn oil
- Concentration of test material in vehicle: 12.5, 25.0, 50.0 mg/mL
- Amount of vehicle (if gavage or dermal): animals in all groups were dosed with the standard volume of 0.1 ml/10 g bodyweight (10 mL/kg bw)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Ugilec 101 was prepared as a solution in corn oil using a high speed mixer.
Duration of treatment / exposure:
2 administrations separated by an interval of 24h.
Frequency of treatment:
daily
Post exposure period:
6 hours
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
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 (recommanded by OECD for this specific test)
- Route of administration: intraperitoneal injection
- Doses / concentrations: 4 mg/kg for 2 days
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
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
at 500 mg/kg bw/d
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:
Phase I (2 animals/sex/dose): 2 administrations from 250 to 9700 mg/kg bw/d (from 500 to 19400 mg/kg bw in total)
Phase II (5 animals/sex/dose): 2 administrations from 500 to 1000 mg/kg bw/d (from 2000 to 4000 mg/kg bw in total)
- Mortality (see Table 1 in the field Remarks on results)
- Clinical signs of toxicity:
After administration of Ugilec 101 at 250 mg/kg bw/d, no toxic reactions were observed.
At 1000 mg/kg bw/d, a toxic reaction consisting of hypopnoea and lethargy was observed 1 hour after the first dose and the symptoms were still present 7 hours later. However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 hour after the second dose and were still present at the time of sacrifice, 5 hours later.
Between 1500 and 9700 mg/kg bw/d, a toxic reaction consisting of hypopnoea, ptosis and pale extremities were observed 1 hour after the first dose. In addition, ataxia and tremors were observed 2 hours later and all the symptoms were still present 4 hours later (8 hours after the first dose). However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 and 3 hours later and 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.
One male treated at 250 mg/kg bw/d (500 mg/kg bw in total) was found dead 24 hours after the first treatment.
One male treated at 500 mg/kg bw/d (1000 mg/kg bw in total) was found dead 2 hours after the second treatment.

- Clinical signs of toxicity:
After administration of Ugilec 101 at dosages of 125 and 250 mg/kg bw/d (250 and 500 mg/kg bw in total, respectively) no toxic reactions were observed.

Dosage of 500 mg/kg bw/d (1000 mg/kg bw in total) produced a toxic reaction consisting of hypopnoea and lethargy which was observed 1 hour after the first dose and the symptoms were still present 7 hours later. However on the morning of the second day (24 hours after the first dose) the symptoms were not observed, but reappeared 1 hour after the second dose and were still present at the time of sacrifice, 5 hours later.

Macrocopic examination at post mortem was not possible for the animal dead in the mid-dose treated group, due to visceral autolysis, and failed to reveal any abnormalities in the high-dose treated dead animal.

- Induction of micronuclei (for Micronucleus assay):
After administration of Ugilec 101 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 101 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 prelimnary toxicity study at the same dose levels.

Table 1 Preliminary toxicity results

Test design

Treatment (2 administrations at 24-h interval)

Mortality ratio

Males

Females

Phase I:
Pilot study

Vehicle

0/2

0/2

TS at 250 mg/kg bw/d

0/2

0/2

TS at 2500 mg/kg bw/d

2/2

2/2

TS at 5000 mg/kg bw/d

2/2

2/2

TS at 7500 mg/kg bw/d

2/2

2/2

TS at 9700 mg/kg bw/d

2/2

2/2

Phase II:
Toxicity assay

Vehicle

0/5

0/5

TS at 1000 mg/kg bw/d

4/5

4/5

TS at 1500 mg/kg bw/d

5/5

3/5

TS at 2000 mg/kg bw/d

5/5

5/5

Table 2 Micronucleus assay results

Treatment  

(2 administrations at 24-h interval)

Sacrifice time
(h)

Mean number of micronucleated cells per 2000 polychromatic erythrocytes per animal

Mean ratio of normochromatic
to polychromatic erythrocytes

Vehicle

30

1.8

1.7

TS at 125

mg/kg bw/d

30

1.9

1.6

TS at 250

mg/kg bw/d

30

1.4

1.4

TS at 500

mg/kg bw/d

30

2.2

1.7

Mitomycin C at 8 mg/kg bw/d

30

105.1

9.0

Conclusions:
Ugilec 101 (benzyltoluene) did not present any in vivo genotoxic activity in the micronucleus test conducted in mice orally gavaged twice at 125, 250 and 500 mg/kg bw.
Executive summary:

The genotoxicity of benzyl toluene (Ugilec 101) 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, 500 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 125, 250, and 500 mg/kg bw/d 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 500 mg/kg bw/d, consisting of hypopnoea, ptosis, ataxia and tremors. 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 benzyltoluene 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. Benzyl toluene was concluded to be negative in the micronucleus test with mice.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From july 26, 1989 to December 13, 1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions
Reason / purpose:
reference to same study
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:
Animals were treated for 4 months; only one dose was tested: no medullar toxicity was elicited at this only tested dose level; only male animals were used; 1000 PCE were scored for micronuclei instead of at least 2000.
GLP compliance:
yes
Type of assay:
micronucleus assay
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 (160 - 207 g)
- Fasting period before study: not reported
- Housing: individually; space allocated: 345 square cm x 17 cm
- Diet: a complete commercial diet 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:
- Vehicle(s)/solvent(s) used: 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:
0 and 500 mg/kg/day
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 administered 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 BT 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).

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.
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 BT 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 BT.

Table 1: Micronucleus assay results

Treatment

Sacrifice time
(d)

Sex

PCE/total erythrocytes
(mean +-/ sd)

Number of micronucleated PCE per 1000 PCE
(mean+-/ sd)

Vehicle control

134 - 143

M

1.55 +/- 0.22

1.95 +/- 0.81

TS at 500 mg/kg

134 - 143

M

1.65 +/- 0.24

2.53 +/- 0.83

Conclusions:
Jarylec BT 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 (Verschuere, 1992) of benzyl toluenes (JARYLEC BT in the report) administered orally (0, 5, 50, and 500 mg/kg/day) to Sprague-Dawley rats, it was determined whether this compound induced micronuclei in bone marrow erythrocytes. 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 group treated at 500 mg/kg/day. No increase in the number of micronucleated, normochromatic or polychromatic cells, and no significant decrease in the PCE/NCE ration were noted. Benzyl toluenes did not present any in vivo genotoxic activity in the micronucleus test after a 4-month treatment in the rat at 500 mg/kg/day.

 

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
supporting 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
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: 25.3 ¿ 31.3 g (males); 21.2 ¿ 27.1 g (females); weight at study repetition: 31.3 ¿ 39.8 g (males); 32.5 ¿ 35.9 g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- 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 resp. 20 days (animals of test repitition)

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: sodium carboxylmethylcellulose, 0.5% in water (Roth GmbH & Co. KG, Karlsruhe, Germany)
- Justification for choice of solvent/vehicle: no data
- Concentration of test material in vehicle: no data
Frequency of treatment:
single intraperitoneal injection of the test article
Post exposure period:
24, 48 and 72 hours
Remarks:
Doses / Concentrations:
600 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:
bone marrow ; poly- and normochromatic erythrocytes, micronucleated cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
On the basis of the results of the range finding study, a dose of 600 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
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: 300, 500, 600 and 800 mg/kg b.w.
- Clinical signs of toxicity in test animals: In the highest-dosed group (800 mg/kg b.ww), all mice (2 male, 2 female) were found dead 4 hours p.a.
Frequent clinical findings in animals treated with 800 mg/kg were reduced activity and respiration rate, abdominal tone, skin turgor, abnormal gait, prone position and piloerection. Slight to moderate piloerection, a slight decrease in activity and skin turgor were the only clinical signs observed in the 600 mg/kg dose group. Animals receiving 500 and 300 mg/kg revealed no relevant abnormal clinical signs.
- Evidence of cytotoxicity in tissue analyzed: not examined


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 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 was slightly increased in the test group (600 mg/kg b.w., both sexes, 24 h). The observed values, however, were within the historical range of control animals and were not considered to be relevant.
- Ratio of PCE/NCE (for Micronucleus assay): The ratio of poly- to normochromatic erythrocytes in the test group (600 mg/kg b.w., females, 48 h) differed significantly (* p < 0.05; U-test) to the corresponding control animals.
- 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 600 mg/kg b.w .were piloerection, abnormal body posture and depressed activity. A ventral position was observed occasionally in some animals. Three animals died in the main study 24 hours p.a.Therefore, an additional nine animals were treated once again. From this group all animals survived and displayed similar symptoms as described above. Only three animals were used for further evaluation.
- Statistical evaluation: see above

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

      Neg. control   test substance 600 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  516.0 ± 44.2  539.4 ± 47.6  446.0 ± 31.9  551.4 ± 119.7  648.8 ± 150.0  389.4 ± 112.5  579.0 ± 98.0 *
   polychromatic 484.0 ± 44.2   460.6 ± 47.6  554.0 ± 31.9  448.6 ± 119.7  351.2 ± 150.0  610.6 ± 112.5  421.0 ± 98.0*
   polychromatic with micronuclei  2.0 ± 1.9  2.6 ± 1.3  2.6 ± 1.1  4.4 ± 2.1  1.8 ± 0.8  1.8 ± 0.8  37.6 ± 15.5**
 ratio of erythrocytes  polychromatic /normochromatic  0.948 ± 0.176  0.868 ± 0.178  1.252 ± 0.170  0.896 ± 0.473  0.626 ± 0.461  1.742 ± 0.757  0.770 ± 0.317*

* = statistically significant (p < 0.05 U-test (Mann-Whitney))

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

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

      Neg. control   test substance 600 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  443.6 ± 59.8  487.2 ± 26.2  393.0 ± 67.6  553.8 ± 158.7  577.0 ± 108.7*  436.6 ± 89.1  461.2 ± 76.0
   polychromatic 556.4 ± 59.8   512.8 ± 26.2  607.0 ± 67.6  446.2 ± 158.7  423.0 ± 108.7  563.4 ± 89.1  538.8 ± 76.0*
   polychromatic with micronuclei  1.0 ± 0.7  2.2 ± 1.8  4.2 ± 1.3  3.6 ± 2.8  2.4 ± 0.5  3.0 ± 1.6  23.2 ± 5.9**
 ratio of erythrocytes  polychromatic /normochromatic  1.292. ± 0.340  1.056 ± 0.110  1.618 ± 0.549  0.964 ± 0.710  0.776 ± 0.283*  1.380 ± 0.569  1.208 ± 0.303

* = statistically significant (p < 0.05 U-test (Mann-Whitney) )

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

Conclusions:
The test substance was considered to be non-mutagenic.
Executive summary:

Benzyltoluene was tested in a mammalian micronucelus test (OECD 474 study, GLP). A single intraperitoneal administration of the test substance at a dose of 600 mg/kg bw to male and female mice produced a slightly significant increase in the frequency of micronuclei in polychromatic erythrocyte cell 24h after administration. According to the historical data of the testing laboratory the mean value of this parameters measured were within the normal range. Under the experimental conditions of this study, the test substance was considered to be non-mutagenic.

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

Additional information

Justification for classification or non-classification

According to EU Regulation (EC) N0. 1272/2008 (CLP), the substance is not classified for genotoxicity.