Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance TBBS was not genotoxic in several bacteria mutation assays and in a mammalian cell mutation assay, the HGPRT assay. Positive responses however were seen in several mouse lymphoma assays in the presence of metabolic activation and in an in vitro chromosome aberration assay in presence of metabolic activation. Because the chemical was non-mutagenic in bacteria and in the HGPRT assay and clastogenic in mammalian cells, the positive response in the mouse lymphoma assays seemed to be derivate from chromosome aberrations. The chemical was negative in the in vivo mouse micronucleus assay (OECD TG 474) tested up to 2000 mg/kg (Durward 2002). Thus, the in vitro clastogenic potential of the test substance was not confirmed in vivo.
In conclusion, the chemical was clastogenic in vitro but not in vivo. This conclusion is in line with the OECD SIDS assessment profile (OECD SIDS 2002).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study, basic data give (abstract and tables)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Principles of method if other than guideline:
N-tert-butyl-2-benzothiazolesulfenamide was tested in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Ames assay
Species / strain / cell type:
other: Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9: 0, 156, 3.13, 6.25, 12.5, 25, 50, 100 mg/plate TA 1537; 0, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 µg/plate TA 98; 0, 15.6,31.3, 62.5, 125, 250, 500, 1000 TA 1535; 0, 39.1, 78.1, 156, 313, 625, 1250, 2500 mg/plate TA 100; 0, 313, 625, 1250, 2500, 5000 mg/plate E. coli WP2 uvr
+S9: 0, 62.5, 125, 250, 500, 1000, 2000 mg/plate; 0, 313, 625, 1250, 2500, 5000: TA 100, TA 1535, TA 98 and E. coli WEP2uvrA
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: -S9: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (TA 100, TA 98, WP2), sodium azide (TA 1535), 9-aminoacridine (TA1537); +S9: 2-aminoanthracene (five strains)
Species / strain:
other: Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537, E. coli WP2 uvrA
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:
valid

The test substance did not induce mutations in the S. thyphimurium and E. coli strans. Toxicity was observed at 25 µg/plate (TA1537), 62.5 µg/plate (TA 1535, TA 98) and 2500 mg/plate (TA100) without an S9 mix, and at 1000 mg/plate (TA 1537) with an S9 mix. Toxicity was not observed in the other cases.

Conclusions:
Interpretation of results: negative
Executive summary:

N-tert-butyl-2-benzothiazolesulfenamide was negative (not mutagenic) in Salmonella typhimurium TA 100, TA 1535, TA 98, TA 1537 and Escherichia coli WP2 uvrA, with or without an exogenous metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline study, basic data give (abstract and tables)
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome aberrations
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9 (continuous treatment): 0, 0.015, 0.03, 0.06 mg/ml; -S9 (short-term treatment): 0, 0.05, 0.1, 0.2 mg/ml; +S9 (short-term treatment): 0, 0.1, 0.2, 0.4 mg/ml.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: mitomycin C; +S9: cyclophosphamide
Species / strain:
mammalian cell line, other: Chinese hamster lung (CHL/IU) cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

Cytotoxicity of the test substance was indicated by a clear reduction of cell confluency at 0.03 (62.5 %) and 0.06 mg/ml (38 %) (24 hour treatment) and at 0.06 mg/ml (60.5%) (48 hour treatment) without metabolic activation. In the short-term treatment (6 hours) without metabolic activation a clear reduction in cell confluency was noted in all treatment groups (65% to 36.5 % compared to control). In the short-term treatment with metabolic activation (6 hours) no decrease in cell confluency was noted in any of the treatment groups (increase up to 222 %). No genotoxic effects of the test substanc were noted without metabolic activation.

Whereas in the short-term treatment with metabolic activation a statistically significant increase of polyploidy and total number of cells with aberrations (TAG) were noted at the highest concentrations (0.2 and 0.4 mg/ml) when compared to the historical control data (no historical control data given).

However, the increase in polyploidy is even low (1.13% and 1.63 %) and the authors stated that the increase is not biologically relevant. Moreover, according to recommendation given in the current OECD guideline 473 (in vitro chromosomal aberration assay) this test system is not designed to measure numerical aberrations and should not routinely be used for that purpose.

The increase in aberrant cells including gaps is significant different (p<0.05) from historical control data (5.0% and 6.0% vs. 2.0% solvent control). The number of cells with aberrations without gaps (TA) is also different from control at the highest concentrations (3.5% and 5.0 % vs. 0.5 % solvent control); however, the authors indicated no statistically significant differences for TA of the highest dose groups compared to the historical control data.

Conclusions:
Interpretation of results: positive with metabolic activation
Executive summary:

N-tert-butylbenzothiazole-2-sulphenamide was positive with metabolic activation in this in vitro mammalian chromosome aberration test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
acceptable documented publication and study report with limitations compared to current guidelines (without metabolic very low background mutation frequencies 8-16 E6 (recommendet greater than 35 E6), no differentiation of small and large mutant-colonies etc.)
Principles of method if other than guideline:
Bionetics method (mouse lymphoma forward mutation assay)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
other: L5178Y
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Up to 15 µg/ml without S9 and up to 60 µg/ml with S9.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: EMS, DMN
Details on test system and experimental conditions:
Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity

The test material induced a small increase in mutant frequency at the tk locus in L5178Y mouse lymphoma cells in the presence of metabolic activation. Mutagenic activity became observable near 20 µg/ml (35.6 % rel. growth, 64.2 x 106 mutant frequency) and 3 -fold increased in mutant frequency (121 x 106 vs. 40.0 x 106 solvent control) were observed for highly toxic treatments with concentrations up to 60 mg/ml (15.1 % rel. growth). Without metabolic activation, the test substance became highly toxic at 15 µg/ml (4.8 % rel. growths) and did not induce an biologically relevant increase in mutant frequency up to the highest concentration evaluated.

Conclusions:
Interpretation of results: positive with metabolic activation
Executive summary:

Mouse lymphoma with L5178Y cells was positive with metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: acceptable documented study report with limitations compared to current guidlines (with and without metabolic very low background mutation frequencies 1-28 E6 (recommendet greater than 35 E6), no differentiation of small and large mutant colonies etc.)
Principles of method if other than guideline:
Bionetics method (mouse lymphoma forward mutation assay).
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
other: L5178Y
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Up to 12.5 µg/ml without S9 and 50 µg/ml with S9.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Details on test system and experimental conditions:
Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks on result:
other: other: L5178Y

The test material Santocure NS BO-78-222 induced an increase in mutation frequency at the tk locus in L5178Y mouse lymphoma cells in the range of high toxicity (relative growths less than 20 %) in the presence of metabolic activation (in two of three trials). Ca. 4.8 to 6.5 -fold increases were obtained with 50 µg/ml (rel. growth 3.3 % to 6.2 % in two trials). In one of three trials a 2.3 -fold increase in the mutant frequency (63.6 x 106vs. 28.1 x 106solvent control) was seen in the range of moderate toxicity (25 µg/ml, 58.2 % rel. growths). Without metabolic activation, highly toxic concentrations up to 17.5 µg/ml were not detectably mutagenic.

The authors concluded, that the test substance is weakly mutagenic with metabolic activation in the mouse lymphoma forward mutation assay.

Conclusions:
Interpretation of results: positive with metabolic activation
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study with acceptable restriction (e.g. no colony sizing performed).
Principles of method if other than guideline:
SRI method (mouse lymphoma assay)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9: 0, 4, 6, 8, 10, 12, 14, 16, 20 µg/ml, +S9: 0, 10, 20, 40, 60, 80, 100, 120, 140 µg/ml
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: EMS, +S9: 3-MCA
Details on test system and experimental conditions:
Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: other: L5178Y

Mutagenesis assays measuring the forward mutation frequency at the thymidine kinase locus in L5178Y mouse lymphoma cells were conducted with the test substance both in the absence and in the presence of an Aroclor-induced metabolic activation preparation (S 9 mix).

Cytotoxicity, indicated by a decrease of the rel. total growth with and without metabolic activation

With metabolic activation: ca. 80 % rel. total growth at 10 µg/ml, at 140 µg/m 7% rel total growth

Without metabolic activation: 10 µg/ml ca. 75 % rel. total growth, 16 µg/ml ca. 25 % and at 20 µg/ml ca.1.5% rel. total growth

Without metabolic activation, no significant increase in the mutation frequency of the cells was observed.

However, in the presence of metabolic activation, concentrations of 10 to 100 µg/ml Santocure NS increased the mutation frequency, in a concentration-dependent manner, above the spontaneous frequency of the control cells. An increase of the mutation frequency above the background frequency was noted in the range of low, mid and higher toxicity in presence of metabolic activation.

Statistic analysis:

The samples treated with the two highest concentrations were not analysed statistically because of their low growth values; the remaining samples (10 to 100 µg/ml) yielded a significant increase in mutation frequency over that of the solvent controls and a significant dose-dependent increase in the response.

Conclusions:
Interpretation of results: positive with metabolic activation
Executive summary:

Mouse lymphoma with L5178Y cells was positive with metabolic activation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, comparable to guideline study with acceptable restriction (e.g. no colony sizing performed)
Principles of method if other than guideline:
SRI method (mouse lymphoma assay)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9: 0, 4, 8, 16, 18, 20, 26, 30, 40 µg/ml, +S9: 0, 20, 40, 60, 80, 90, 100, 120, 160 µg/ml
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: -S9: EMS, +S9: 3-MCA
Details on test system and experimental conditions:
Mouse lymphoma assay
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: L5178Y

Without Metabolic activation:

In the absence of metabolic activation, recrystallized Santocure NS was very toxic at concentrations greater than 20 µg/ml. At the lowest concentration tested, 4 µg/ml the mean rel. total growth was 93.9%.The mean mutation frequencies of the three highest concentrations with mean rel. total growth values of at least 10 % were significantly greater (p< 0.01) than that of the solvent control. The linear component of the concentration (dose)- response relationship was statistically significant (p< 0.0001) and exhibited a postive slope for test conentrations with mean relative total growth values of at least 10 %.

With metabolic acitivation:

with metabolic activation, recrystallized Santocure NS was very toxic at concentrations greater than 100 µg/ml. At 20 µg/ml, the mean rel. total growth value was 76.2%. The mean mutation frequencies of three od the four highest concentrations with mean relative total growth values of at least 10 % were significantly greater (p< 0.01) than that of the solvent control. The linear component of the concentration (dose)- response relationship was statistically significant (p< 0.0003) and exhibited a postive slope for test concentrations with mean relative total growth value of at least 10 %.

The combined results indicated a positive mutagenic response for the test substance in the tk locus in L5178Y mouse lymphoma cells, both in the absence and in the presence of the metabolic activation system.

Conclusions:
Interpretation of results: positive
Executive summary:

Mouse lymphoma with L5178Y cells was positive with metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Groups, each of seven mice, were dosed once via the intraperitoneal route with the test substance at 500, 100 or 2000 mg/kg bw. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg bw was killed after 48 hours.
Immediately following termination (i.e. 24 or 48 hours after dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
CD-1
Sex:
male
Route of administration:
intraperitoneal
Duration of treatment / exposure:
24 and 48 h
Frequency of treatment:
once
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:

No. of animals per sex per dose:
7 males/ dose
Control animals:
yes, concurrent vehicle
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid

Range-finding study:

In animals dosed with test material, at maximum recommended dose level of 2000 mg/kg bw, via the oral and intraperitoneal route no premature deaths were recorded. One animal dosed via the intraperitoneal route demonstrated one clinical sign, diuresis.

The test material showed no marked differences in its toxicity to male or female mice; it was therefore considered from the authors to be acceptable to use males only for the main study. The intraperitoneal route was selected for use in the main study in an attempt to maximize exposure of the animals to the test substance. The maximum recommended dose (MRD) of the test substance, 2000 mg/kg bw, was selected for use in the main study, with 1000 and 500 mg/kg bw as the lower dose levels.

Micronucleus study:

Mortality: none during the study

Clinical signs: Clinical signs were observed in animals dosed with test material at and above 500 mg/kg and included as follows: hunched posture, pilo-erection, lethargy and ptosis. The animals appeared to be more sensitive to the test substance in the main-study than observed in the preliminary range-finding study.

Effect on PCE/NCE ratio: a statistically significant decrease in the PCE/NCE ratio was seen in the 48 -hour 2000 mg/kg and 24 -hour 500 mg/kg test material groups when compared to their concurrent vehicle control groups. The PCE/NCE ratios for the 1000 and 2000 mg/kg 24 -hour test material dose groups were also lower than their concurrent vehicle control. These, accompanied by the observation of clinical signs, were taken to indicate that systemic absorption and adequate exposure of the bone marrow had been achieved.

Genotoxic effects: Negative.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

The positive control group showed a market increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Summary of group mean data

 Treatment group  Number of PCE with micronuclei per 2000 PCE    PCE/NCE ratio  
   Group mean  SD  Group mean  SD
 Vehicle control, 48 h sampling time  2.0 1.2  0.88  0.21 
 Vehicle control, 24 h sampling time  2.6 2.3  0.85  0.26 
Positive control, 24 h sampling time   34.00** 18.3 1.56  0.63 
 2000 mg/kg, 48 h sampling time  1.4 1.3  0.59*  0.25 
 2000 mg/kg, 24 h sampling time  2.4  2.6  0.71  0.38
 1000 mg/kg, 24 h sampling time  2.1  2.3  0.62  0.32
 500 mg/kg, 24 h sampling time  1.6  1.4  0.53*  0.27

* p< 0.05

** p< 0.001

Conclusions:
Interpretation of results: negative
Executive summary:

Groups, each of seven mice, were dosed once via the intraperitoneal route with the test substance at 500, 100 or 2000 mg/kg bw. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with 2000 mg/kg bw was killed after 48 hours.

Immediately following termination (i.e. 24 or 48 hours after dosing), both femurs were were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored.

There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

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

Additional information

In vitro data

The mutagenic potential in bacteria of the test substance TBBS was evaluated in a GLP and OECD guideline study.

Here, the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were used. Treatment by the pre-incubation method was done for doses up to 5000 µg/ plate or even lower if toxicity was indicated. The tester strains were evaluated with and without metabolic activation. In this study no biologically relevant and dose dependent increases in revertants was observed in any of the tester strains evaluated with and without metabolic activation (MHWJ 1997).

In addition, in earlier bacterial mutation assays the test substance also indicated a non-mutagenic potential (Monsanto 1976, Hinderer 1983).

The test substance was evaluated in an in vitro chromosome assay (TG 473) with CHL-cells (MHWJ 1997). The cells were treated in a short-term treatment with and without metabolic activation and in continue treatments for 24 and 48 hours without metabolic activation. Cytotoxicity was noted in short- and long-term treatments without metabolic activation, indicated by a reduction of the cell confluence. No cytotoxicity of the test substance was noted in the presence of metabolic activation up to the highest concentration evaluated (0.4 mg/ml). No biologically relevant increase in aberrant cells or polyploidy was noted without metabolic activation. Whereas in the short-term treatment with metabolic activation a statistically significant increase of polyploidy and total number of cells with aberrations (TAG) were noted at the highest concentrations (0.2 and 0.4 mg/ml) when compared to the historical control data. However, the increase in polyploidy was even low (1.13% and 1.63 %) and the authors stated that the increase is not biologically relevant. The increase in aberrant cells including gaps was significant different (p<0.05) from historical control data (5.0% and 6.0% vs. 2.0% concurrent solvent control). The number of cells with aberrations without gaps (TA) was also different from the solvent control at the highest concentrations (3.5% and 5.0 % vs. 0.5 % concurrent solvent control). Thus, based on the findings of this study, the test substance reveals a weak but statistically significant mutagenic effect in presence of metabolic activation.

No increases in aberrant cells were detected in an earlier in vitro chromosome assay (Hinderer 1983). However, this study reveals limitations concerning the test design which limited the validity of the study.

A mutagenic response in presence of metabolic activation was also found in several mouse lymphoma forward mutation studies mainly in the presence of metabolic activation (Monsanto Co.1979, Monsanto 1982, 1982b, Hinderer 1983). Whereas, in a limited documented HGPRT assay with CHO cells (only secondary literature) no mutagenicity was indicated (ECB IUCLID 2000).

In vivo data

In an in vivo micronucleus assay (OECD TG 474) with CD-1 male mice no genotoxic effects of the test substance TBBS was observed. Male CD-1 mice were treated once ip. with 0, 500, 1000 or 2000 mg/kg test substance. No mortality occurred in any of the treated animals. Clinical signs like pilo-erection, lethargy and ptosis were noted in animals of the 500 mg/kg treatment group and higher. A decrease of the PCE/NCE ratio was noted in all treated animals compared to the concurrent vehicle control. Moreover, the decrease in the PCE/NCE ratio was statistically significant decrease in the 48-hour 2000 mg/kg (0.59) and 24-hour 500 mg/kg (0.53) groups when compared to the concurrent vehicle controls (0.88, 0.85). The indication of clinical signs revealed systemic availability and the decrease in the PCE/NCE ratio indicated that the target tissue was reached by the test substance, and thus reveals the validity of the test system. No statistically significant increases in the frequency of micronucleated polychromatic erythrocytes (PCE) were noted in any of the treated animals compared to the concurrent vehicle control. Whereas, the positive control group showed a market increase in the incidence of micronucleated polychromatic erythrocytes, hence confirming the sensitivity of the test system to the known mutagenic activity of cyclophosphamide under the conditions of the test (Durward 2002).

In conclusion, the test substance TBBS was negative in an in vivo micronucleus assay with CD-1 mice.


Justification for selection of genetic toxicity endpoint
Several in-vitro assays (Ames tests, chromosome aberrations test, mouse lymphoma assays and a HGPRT assay in CHO cells) and an in-vivo assay are available.

Short description of key information:
As discussed in the OECD SIDS (2003), the test substance TBBS was not genotoxic in several bacteria mutation assays and in a mammalian cell mutation assay, the HGPRT assay. Positive responses however were seen in several mouse lymphoma assays in the presence of metabolic activation and in an in vitro chromosome aberration assay in presence of metabolic activation. Because the chemical was non-mutagenic in bacteria and in the HGPRT assay and clastogenic in mammalian cells, the positive response in the mouse lymphoma assays seemed to be derivate from chromosome aberrations. The chemical was negative in the in vivo mouse micronucleus assay (OECD TG 474) tested up to 2000 mg/kg (Durward 2002). Thus, the in vitro clastogenic potential of the test substance was not confirmed in vivo.
In conclusion, the chemical was clastogenic in vitro but not in vivo. This conclusion is in line with the OECD SIDS assessment profile (OECD SIDS 2002).

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

TBBS was not genotoxic in several bacteria mutation assays and in a mammalian cell mutation assay, the HGPRT assay. Positive responses however were seen in several mouse lymphoma assays in the presence of metabolic activation and in an in vitro chromosome aberration assay in presence of metabolic activation. Because the chemical was non-mutagenic in bacteria and in the HGPRT assay and clastogenic in mammalian cells, the positive response in the mouse lymphoma assays seemed to be derivate from chromosome aberrations. The chemical was negative in the in vivo mouse micronucleus assay (OECD TG 474) tested up to 2000 mg/kg (Durward 2002). Thus, the in vitro clastogenic potential of the test substance was not confirmed in vivo.

In conclusion, the chemical was clastogenic in vitro but not in vivo. This conclusion is in line with the OECD SIDS assessment profile (OECD SIDS 2002).

According to CLP classification criteria (Regulation (EC) No 1272/2008) a classification is not proposed.