1,2-benzisothiazol-3(2H)-one

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

Purity: 77.4% 1,2-Benzisothiazol-3(2H)-one (BIT)
Batch number Bx.L1001

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction

Test concentrations with justification for top dose:

Test 1: 0.1 to 3.2 µg/ml and 0.2 to 6.4 µg/ml without and with metabolic activation, respectively.
Test 2: 0.8 to 6.4 µg/ml and 1.6 to 12.8 µg/ml without and with metabolic activation, respectively.

Details on test system and experimental conditions:

IUCLID4 Type: Mouse lymphoma assay

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Materials and methods
BIT was assayed for mutation at the hprt locus (6-thioguanine resistance) in mouse lymphoma cells using a fluctuation protocol (guideline compliance with OECD 476 (1997) and EC B.17 (2000)). The study consisted of cytotoxicity range-finding experiments followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9).
The initial cytotoxicity range-finding experiment with and without metabolic activation was performed in the concentration range 46.88 to 1512 µg/mL. Since complete toxicity was observed at all concentrations, a second range-finding experiment was performed with and without metabolic activation in the range 0.3906 to 50 µg/mL. On the basis of the results from this second range-finding experiment, Experiment 1 of the main test was performed in the concentration range 0.1 to 3.2 µg/mL in the absence of metabolic activation and in the concentration range 0.2 to 6.4 µg/mL in the presence of metabolic activation. The concentration range was extended in Experiment 2 of the main test to 0.2 to 6.4 µg/mL in the absence of metabolic activation and to 0.4 to 12.8 µg/mL in the presence of metabolic activation.
Cultures with BIT, 4 Nitroquinoline 1-oxide (NQO, positive control in the absence of metabolic activation) or Benzo(a)pyrene (BP, positive control in the presence of metabolic activation) were maintained in flasks for a period of 7 days during which the HPRT mutation would be expressed.
Mutant frequency was assessed for statistical significance. The experiment was considered valid if the mutant frequencies in the solvent control cultures fell within the normal range (not more than three times the historical mean value) and at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the mutant frequencies was greater than half the historical mean value).
The test substance was considered to be mutagenic if the assay was valid, the mutant frequency of one or more doses was significantly greater than that of the solvent control, and there was a significant dose relationship as indicated by the linear trend analysis and if these effects were reproducible.

Results and discussion
In Experiment 1, the highest dose selected in the presence of S-9 (6.4 µg/mL) could not be analysed due to excessive contamination observed on the mutant plates and the second highest dose tested (4.8 µg/mL) was considered too toxic for selection to determine viability and 6-thioguanine (6TG) resistance (extreme toxicity in one of the replicate cultures was observed). The highest dose analysed in both the absence and presence of S-9 was therefore 3.2 µg/mL, with relative survival being 61% and 44%, respectively. No dose of ideal toxicity (10-20% relative survival) was achieved in the absence or presence of S-9. This was unexpected, based on the results of the cytotoxicity range-finding experiment. However, adequate, dose-related toxicity was demonstrated in the absence and presence of S-9 in Experiment 2, therefore this was not considered to have affected the integrity of the study in any way.
In Experiment 2, the highest doses analysed were 4.8 µg/mL in the absence of S-9 and 6.4 µg/mL in the presence of S 9, with relative survival being 9% and 10%, respectively.
Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of metabolic activation. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-Nitroquinoline 1-oxide (without metabolic activation) and Benzo(a)pyrene (with metabolic activation). The study was therefore considered valid.
No statistically significant increases in mutant frequency were observed following treatment with BIT at any dose level analysed, in the absence or presence of metabolic activation in Experiment 1, or in the absence of metabolic activation in Experiment 2.
A small but statistically significant increase in mutant frequency was observed at the highest dose analysed in the presence of metabolic activation in Experiment 2 (6.4 µg/mL), compared to the concurrent solvent controls, and a linear trend was observed. The mutant frequency observed at this dose was, however, similar to the historical mean solvent control mutant frequency. Furthermore, the statistically significant increase in mutant frequency was observed at a highly toxic dose, yielding only 10% relative survival. This increase in mutant frequency was therefore considered of little or no biological significance.

Conclusion
Under the conditions employed in this study, BIT did not show conclusive evidence of mutagenic activity.

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the substance in mammalian cells according to OECD Guideline 476. The test substance was assayed for mutation at the hprt locus (6-thioguanine resistance) in mouse lymphoma cells. The study consisted of cytotoxicity range-finding experiments followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The initial cytotoxicity range-finding experiment with and without metabolic activation was performed in the concentration range 46.88 to 1512 µg/mL.  Since complete toxicity was observed at all concentrations, a second range-finding experiment was performed with and without metabolic activation in the range 0.3906 to 50 µg/mL. On the basis of the results from this second range-finding experiment, Experiment 1 of the main test was performed in the concentration range 0.1 to 3.2 µg/mL in the absence of metabolic activation and in the concentration range 0.2 to 6.4 µg/mL in the presence of metabolic activation.  The concentration range was extended in Experiment 2 of the main test to 0.2 to 6.4 µg/mL in the absence of metabolic activation and to 0.4 to 12.8 µg/mL in the presence of metabolic activation. 4 Nitroquinoline 1 -oxide (in the absence of metabolic activation) or Benzo(a)pyrene (in the presence of metabolic activation) were used as the positive controls in this study. Mutant frequency was assessed for statistical significance. In experiment 1, the highest dose selected in the presence of S-9 (6.4 µg/mL) could not be analysed due to excessive contamination observed on the mutant plates and the second highest dose tested (4.8 µg/mL) was considered too toxic for selection to determine viability and 6 -thioguanine (6TG) resistance. The highest dose analysed in both the absence and presence of S-9 was therefore 3.2 µg/mL, with relative survival being 61% and 44%, respectively. No dose of ideal toxicity (10-20% relative survival) was achieved in the absence or presence of S-9. However, adequate dose-related toxicity was demonstrated in the absence and presence of S-9 in Experiment 2, therefore this was not considered to have affected the integrity of the study in any way. In experiment 2, the highest doses analysed were 4.8 µg/mL in the absence of S-9 and 6.4 µg/mL in the presence of S 9, with relative survival being 9% and 10%, respectively. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive controls. The study was therefore considered valid. No statistically significant increases in mutant frequency were observed following treatment at any of the non-toxic doses analysed. Under the study conditions, the substance was not considered to be mutagenic in mouse lymphoma cells (Lloyd, 2003).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study can be considered to be essentially compatible with the current OECD Guideline 471 with a few exceptions:

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Purity: 73.4%
Proxel Press Paste
Batch number: ADH374793 Bx973

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

Test concentrations with justification for top dose:

With metabolic activation: 200 to 0.32 µg Proxel press paste/plate, Without metabolic activation: 80 to 0.064 µg Proxel press paste/plate

Details on test system and experimental conditions:

IUCLID4 Type: Ames test

Key result

Species / strain:

other: S. typhimurium strains TA98, TA100, TA1535, TA1537, TA1538

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

Conclusions:

Materials and methods
PROXEL Press Paste was evaluated in the bacterial mutagenicity assay, using five strains of Salmonella typhimurium (TA1535, TAl537, TA1538, TA98 and TA100). The study is considered to be compatible with OECD Guideline 471 (study initiated in 1988 and performed according to OECD Guideline 471, 1983).
A preliminary dose range finding test was performed with strain TA100 in the absence and presence of metabolic activation in the range 5000 to 1.6 µg/plate followed by a preliminary test in the range 200 to 10 µg/plate in strain TA100 only. Given the cytotoxicity of PROXEL Press Paste observed in the absence and presence of metabolic activation (80 µg/plate and 200 µg/plate, respectively) the main study was conducted with all strains at concentrations of 80 to 0.064 µg/plate without metabolic activation and of 200 to 0.32 µg/plate with metabolic activation.
Each plate was prepared by the ‘plate incorporation procedure’ where the appropriate components were added to a bijou bottle. These components were the appropriate bacterial strain; S-9 mix (for tests with metabolic activation) or buffer (for tests without metabolic activation); appropriate concentration of PROXEL Press Paste; appropriate chemical for positive controls or DMSO for negative controls; and top agar (10 mL histidine/bioton stock solution:100 mL agar (v/v)). The contents of the bijou bottle were poured immediately onto the surface of a prepared Vogel Bonner plate, allowed to gel and incubated inverted at 37 °C for approximately 66 hours in the dark.
After incubation revertant colonies were counted using an automated colony counter (AMS 40-10 Image Analyser). The test data were analysed for validity and for any reproducible dose related increase in revertant colonies. Statistical analysis was performed using a one tailed Student’s t-test.


Results and discussion

PROXEL Press Paste induced significant cytotoxicity in all strains tested at concentrations of 80 µg/plate in the absence of metabolic activation and 200 µg/plate in the presence of metabolic activation.
In the absence of metabolic activation. PROXEL Press Paste did not induce any significant, reproducible increases in the observed numbers of revertant colonies in strains TA100, TA1535 or TA1538. There was one result in each test performed with strain TA98 in the absence of metabolic activation which could be considered indicative of a possible effect. These data however are not considered to indicate a mutagenic response since the results are not statistically significant and confined to only one dose level in each test. In one experiment performed with TA1537 without metabolic activation there were three results which could be considered indicative of a possible effect. These data are also not considered to indicate a mutagenic response since the results are not statistically significant and confined to only one test.
In the presence of metabolic activation. PROXEL Press Paste did not induce any significant, reproducible increases in the observed numbers of revertant colonies in strains TA100 or TA1535. In both experiments with metabolic activation, statistically significant responses were observed in strain TA98, reaching a maximum response of 1.6 × background in each case. However these effects were not reproducible in two further experiments with TA98 with metabolic activation. A statistically significant response to PROXEL Press Paste was observed with strains TA1537 and TA1538 with metabolic activation in the second experiment but not in the first. Although the maximum response observed exceeded 2 × background in both strains, no reproducible effects were obtained in one further experiment with TA1537 or in two experiments with TA1538. This lack of reproducibility indicates that the observed effects, in the presence of metabolic activation, in these three strains are not due to compound induced mutations.
The positive control data for each strain tested showed evidence of a mutagenic response which was dose related in the absence and presence of metabolic activation. The chemicals used for positive control samples therefore induced the expected response, indicating that all strains were behaving appropriately for this reverse mutation assay.
It can be concluded that under the conditions of this assay there was a non-mutagenic response to PROXEL Press Paste when tested to limit doses of 200 µg/plate (with metabolic activation) and 80 µg/plate (without metabolic activation ), at which concentrations significant toxicity was observed in each case.
[It should be noted that in a study performed by the same laboratory (Callander R.D; 1988; Central Toxicological Laboratory Report No. CTL/P/2056) using the same strains of S. typhimurium and dose ranges of 300 to 4.0 µg/plate, 250 to 0.8 µg/plate (both with metabolic activation) and 200 to 0.8 µg/plate (without metabolic activation), PROXEL Press Paste induced reproducible, dose-related, significant increases in the observed numbers of revertant colonies in strains TA1538 and TA98 in the presence of metabolic activation. These effects were not observed in strains TA1535, TA1537 or TA100 in the presence of metabolic activation. In the absence of metabolic activation, PROXEL Press Paste did not induce any significant reproducible increases in the observed numbers of revertant colonies in any of the five tester strains used.
In another study (May K.; 1995; Pharmaco-LSR Ltd., Report No. 94/NLL051/0788) which tested the mutagenic effects of Nipacide BIT at dose ranges of 100 to 1.0 µg/plate (with and without metabolic activation) on strains TA98, TA100, TA1535 and TA1537, there was no evidence of mutagenic activity under the conditions of the test. Trueman R.W, 1979 (Central Toxicological Laboratory Report No. CTL/R/494) tested strains TA1535, TA1537, TA1538, TA100 and TA98 at a dose range of 2500 to 0.32 µg/plate (with and without metabolic activation) and there was no mutagenic effect observed in the presence or absence of metabolic activation.
When the data from May (1995) and Trueman (1979) are considered together with the Callander (1989) data (presented in this summary) it can be concluded that the presence of 1,2-Benzisothiazolin-3-one is unlikely to evoke mutagenic activity in this type of assay. The positive result observed in Callander (1988) was not observed in three other studies performed under similar conditions.]

Conclusion
Under the conditions of this assay there was a non-mutagenic response to PROXEL Press Paste when tested to limit doses of 200 µg/plate (with metabolic activation) and 80 µg/plate (without metabolic activation ), at which concentrations significant toxicity was observed in each case.
Reliability 2

Deficiencies

The study can be considered to be essentially compatible with the current OECD Guideline 471 with the following exceptions:
The chemicals used for the positive controls in the absence of metabolic activation were N-Methyl-N'-nitro-N-nitrosoguanidine (TA100 and TA1535), Acridine mutagen (TA1537), 4-Nitro-o-phenylenediamine (TA1538) and Daunomycin HCl (TA98). The current guideline recommends Sodium azide (TA100 and TA1535), 9 Aminoacridine (TA1537) and 2 Nitrofluorine (TA98) in the absence of metabolic activation. The use of some currently non-standard positive control chemicals is not considered to affect the reliability of the generated results since the positive control data for each strain tested showed evidence of a mutagenic response which was dose related in the absence and presence of metabolic activation.
The growth phase and cell density of the cultures used was not specifically reported, however guideline compliance (i.e. late exponential or early stationary phase of growth and approximate cell density of 109 cells/mL) is claimed.
Five strains of S. typhimurium are recommended in the test guideline. Although five strains were tested, only four of the recommended strains (TA98, TA100, TA1535, TA1537) were tested, along with TA1538 (TA102, capable of detecting certain mutagens which the other strains cannot, was not tested). As testing of PROXEL Press Paste in the in vitro mammalian cell (mouse lymphoma) gene mutation test and in two in vivo (micronucleus and UDS) assays has generated negative results, the absence of in vitro gene mutation testing in S. typhimurium TA102 is not considered critical overall in the context of the genotoxicity database.

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the substance according to a method equivalent to OECD Guideline 471. The test substance was evaluated in the bacterial mutagenicity assay, using five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100). Based on the cytotoxicity of the test substance in the absence and presence of metabolic activation (80 µg/plate and 200 µg/plate, respectively) in a range-finding study, the main assay was conducted at concentrations from 0.064 to 80 µg/plate without metabolic activation and from 0.32 to 200 µg/plate with metabolic activation. Each plate was prepared by the plate incorporation procedure. After incubation of the plates, the revertant colonies were counted using an automated colony counter (AMS 40-10 Image Analyser).  The test data were analysed for validity and for any reproducible dose related increase in revertant colonies.  Statistical analysis was performed using a one tailed Student’s t-test. The test substance induced significant cytotoxicity in all strains at 80 µg/plate in the absence of metabolic activation and 200 µg/plate in the presence of metabolic activation. The positive control data for each strain tested showed evidence of a mutagenic response which was dose related in the absence and presence of metabolic activation. Under the study conditions, the substance was not considered to be mutagenic in S. typhimurium strains in the presence and absence of metabolic activation (Callander, 1989).

[It should be noted that in a study performed by the same laboratory (Callander R.D; 1988; Central Toxicological Laboratory Report No. CTL/P/2056) using the same strains of S. typhimurium and dose ranges of 300 to 4.0 µg/plate, 250 to 0.8 µg/plate (both with metabolic activation) and 200 to 0.8 µg/plate (without metabolic activation), PROXEL Press Paste induced reproducible, dose-related, significant increases in the observed numbers of revertant colonies in strains TA1538 and TA98 in the presence of metabolic activation.  These effects were not observed in strains TA1535, TA1537 or TA100 in the presence of metabolic activation.  In the absence of metabolic activation, PROXEL Press Paste did not induce any significant reproducible increases in the observed numbers of revertant colonies in any of the five tester strains used.    

In another study (May K.; 1995; Pharmaco-LSR Ltd., Report No. 94/NLL051/0788) which tested the mutagenic effects of Nipacide BIT at dose ranges of 100 to 1.0 µg/plate (with and without metabolic activation) on strains TA98, TA100, TA1535 and TA1537, there was no evidence of mutagenic activity under the conditions of the test.  Trueman R.W, 1979 (Central Toxicological Laboratory Report No. CTL/R/494) tested strains TA1535, TA1537, TA1538, TA100 and TA98 at a dose range of 2500 to 0.32 µg/plate (with and without metabolic activation) and there was no mutagenic effect observed in the presence or absence of metabolic activation.

When the data from May (1995) and Trueman (1979) are considered together with the Callander (1989) data (presented in this summary) it can be concluded that the presence of 1,2-Benzisothiazolin-3-one is unlikely to evoke mutagenic activity in this type of assay.  The positive result observed in Callander (1988) was not observed in three other studies performed under similar conditions.]

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Nipacide BIT
1,2-Benzisothiazol-3(2H)-one (BIT ) purity of 98.8%
Batch A 88

Species / strain / cell type:

other: human lymphocytes

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Preliminary toxicity test: 8 to 5000 µg/ml; First cytogenic test: 1 to 32 µg/ml; Repeat First Cytogenic Test (48 hour, metabolic activation only) 24 to 40 µg/ml and Second cytogenic test: 2 to 16 µg/ml Nipacide BIT.

Key result

Species / strain:

lymphocytes: human

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

Additional information on results:

It is concluded that Nipacide BIT, under the conditions of test, was clastogenic in the absence of S-9 mix at concentrations showing moderate levels of toxicity.

Conclusions:

Materials and methods
The effects on chromosomal structure of exposure to Nipacide BIT were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix). In the absence of metabolic activation, cells were exposed continuously for 21 and/or 45 hours; with metabolic activation, exposure was limited to three hours and cells were harvested 18 or 42 hours later (again resulting in sampling times of 21 or 45 hours).
Treatments were established by the addition of test solutions (in dimethyl sulphoxide; DMSO) to 48-hour cultures established from whole, human blood. Cell division was arrested by the addition of the spindle poison, colcemid three hours before the cells were harvested and slides were then prepared for microscopic analysis.
Mitotic indices were calculated for each culture, these were based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations.
A preliminary test was performed to investigate the toxicity of Nipacide BIT over the concentration range of 8 to 5000 µg/mL to dividing lymphocytes. Exposure to Nipacide BIT in the absence and presence of metabolic activation induced significant cytotoxicity at 40 µg/mL. Subsequently the first cytogenetic test was performed using Nipacide BIT concentrations in the range 1 to 32 µg/mL, to cover the appropriate range of toxicity.
After consideration of results from the first cytogenetic test, the Nipacide BIT concentrations tested in the second cytogenetic test, at the 21 hour sampling time only, were in the range 2 to 16 µg/mL.
The main tests also incorporated solvent (DMSO) and positive (cyclophosphamide and chlorambucil) control cultures. Cyclophosphamide is a known clastogen requiring biotransformation to achieve optimum activity; chlorambucil is a direct-acting clastogen. All control and test exposures were established in duplicate cultures.

Results and discussion
Exposure to Nipacide BIT in the absence of metabolic activation induced significant cytotoxicity at 32 µg/mL (observed in all of the main toxicity tests). In the presence of metabolic activation, significant cytotoxicity was observed at 40 µg/mL Nipacide BIT in the preliminary tests. There was evidence of significant cytotoxicity in one replicate at 24 µg/mL and one replicate at 32 µg/mL in one of the main toxicity tests after the 45 hour sampling time.
In the absence of metabolic activation, treatment with Nipacide BIT at the highest concentrations selected for chromosomal analysis produced statistically significant increases in the frequency of metaphases with aberrant chromosomes, compared to solvent control values (p<0.001, both including and excluding gap-type aberrations) at the 21 hour sampling time. The increases exceeded the historical solvent control range at the testing laboratory and were reproducible. Statistically significant increases were also observed in cultures treated in the presence of S-9 mix, but the frequencies of aberrant metaphases did not exceed the historical solvent control range.
The known clastogens, cyclophosphamide and chlorambucil, induced significant increases in the frequency of metaphases with aberrant chromosomes, compared to the solvent control values, in both cytogenetic tests (p<0.001 in all cases), thus demonstrating the sensitivity of the test procedure, and the metabolic activity of the S-9 mix employed.
It is concluded that Nipacide BIT, under the conditions of test, was clastogenic in the absence of S-9 mix at concentrations showing moderate levels of toxicity.

Conclusion
It is concluded that Nipacide BIT, under the conditions of test, was clastogenic in the absence of S-9 mix at concentrations showing moderate levels of toxicity.

Executive summary:

A study was conducted to determine the in vitro genotoxic potential of the substance according to OECD Guideline 473. The effects of the test substance on chromosomal structure was investigated in cultured human lymphocytes with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix).  In the absence of metabolic activation, cells were exposed continuously for 21 and/or 45 hours; with metabolic activation, exposure was limited to three hours and cells were harvested 18 or 42 hours later (again resulting in sampling times of 21 or 45 hours). Treatments were established by the addition of test solutions in dimethyl sulphoxide at 1 to 32  µg/mL (experiment 1) or at  24 to 40 µg/mL (repeat experiment) and at 2 to 16 µg/mL (experiment 2) to 48-hour cultures of whole, human blood. Cell division was arrested by the addition colcemid three hours before the cells were harvested and slides were then prepared for microscopic analysis. Mitotic indices were calculated for each culture based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations. In the absence of metabolic activation significant cytotoxicity was observed at 32 µg/mL and in the presence of metabolic activation, cytotoxicity was observed at 24 or 32 µg/mL. In the absence of metabolic activation, the highest concentrations selected for chromosomal analysis produced statistically significant increases in the frequency of chromosomal aberrations at the 21 hour sampling time. The increases exceeded the historical solvent control range at the testing laboratory and were reproducible. The positive and negative controls were considered valid. Under the study conditions, the substance was considered to be genotoxic in the chromosomal aberration assay with cultured human lymphocytes (Edwards, 1995).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

unscheduled DNA synthesis

Specific details on test material used for the study:

Proxel Press Paste
Batch BX103

The Test Substance employed was pre-dried technical grade active substance.
Purity 93.1%

Species:

rat

Strain:

other: Han Wistar rats

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

one dose administered by oral gavage

Remarks:

Doses / Concentrations:
560 mg/kg and 1400 mg/kg

Control animals:

yes, concurrent vehicle

Positive control(s):

• Acetamidofluorene (2-AAF) suspended in corn oil at a concentration of 7.5 mg/L
• Dimethylnitrosamine (DMN) dissolved in purified water at a concentration of 1.0 mg/L

Tissues and cell types examined:

100 hepatocytes were analysed per animal, where possible using two out of three slides in each case.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

When treated orally once with PROXEL Press Paste at doses up to 1400 mg/kg, male rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 12-14 or 2-4 hours after dosing.  It is concluded that PROXEL Press Paste had no genotoxic activity detectable in this test system under the experimental conditions employed.

Conclusions:

Materials and methods
In an initial toxicity range-finder study, groups of three male rats were dosed once with 700, 1000. 1400 and 2000 mg/kg PROXEL Press Paste (BIT). Mortalities were seen at 2000 mg/kg and therefore a dose of 1400 mg/kg was considered representative of a maximum tolerated dose.
Groups of at least three male rats were treated once with the solvent 0.5% methyl cellulose; PROXEL Press Paste (BIT) at 560 mg/kg or 1400 mg/kg; or the required positive control, by oral gavage, at a dose volume of 10 mL/kg. The positive controls used were 75 mg/kg 2 acetamidofluorene (2-AAF) suspended in corn oil (12-14 hour experiment) and 10 mg/kg dimethylnitrosamine (DMN) dissolved in purified water (2-4 hour experiment).
Approximately 12-14 hours (Experiment 1) or 2-4 hours (Experiment 2) after dosing, animals were killed and their livers perfused with collagenase to provide a primary culture of hepatocytes. Cultures were made from three animals in each dose group (or in the case of the 1400 mg/kg dose group in the 12-14 hour experiment, from the two surviving animals) and were treated with [3H] thymidine. Six slides from each animal were prepared with fixed hepatocytes and of these three were dipped in photographic emulsion to prepare autoradiograms. Slides were examined microscopically after development of the emulsion and staining, and the net grain count (NNG), the number of grains present in the nucleus minus the mean number of grains in three equivalent areas of cytoplasm, was determined for one hundred cells with normal morphology (from two of the three slides prepared). For each slide, animal and dose point, the population average NNG and percentage of cells responding or in repair (NNG of ≥ 5) was calculated.
The data were evaluated for indication of PROXEL Press Paste induced UDS. The criteria for a positive result were if the PROXEL Press Paste (at any dose at either post dose time point) yielded group mean NNG values of zero or above and ≥ 20% of cells had mean NNG values of ≥ 5. The data would also be indicative of a positive result if an increase above the solvent control levels was seen in both NNG and the percentage in repair.

Results and discussion
In the preliminary study during the 2 day post-dose observation period piloerection, weight loss, lethargy and abnormal breathing (1000 mg/kg dose level only) were observed at 700, 1000 and 1400 mg/kg dose levels. At 2000 mg/kg, piloerection, eye closure, abnormal breathing and lethargy were observed. One animal was killed in extremis shortly after dosing at 2000 mg/kg and one animal from this dose group was found dead one day after dosing. In the remaining animal some weight loss was observed.
As mortalities were seen at 2000 mg/kg, a dose of 1400 mg/kg was considered representative of a maximum tolerated dose. The maximum dose level of 1400 mg/kg was therefore selected for the main study which was tested together with a dose rate of 560 mg/kg.
In the main study, clinical signs of piloerection and lethargy were observed at the top dose tested, 1400 mg/kg, in both experiments. On the day after dosing with 1400 mg/kg PROXEL Press Paste in the 12-14 hour experiment, two animals (out of four animals) were found dead. No clinical signs were observed in the 560 mg/kg dose group.
Negative (vehicle) control animals gave group mean NNG values of 2.3 and 0 in experiments 1 and 2, respectively. These values did not exceed the upper limit of the historical negative control range and only 0 to 0.3% cells in experiments 1 and 2 were in repair. Group mean NNG values were increased by 2-AAP and DMN treatment to ≥ 5 and more than 50% cells found to be in repair. The vehicle control NNG value was consistent with both published and historical control data, and the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid.
Treatment with PROXEL Press Paste at doses up to 1400 mg/kg yielded NNG values less than zero, producing group mean NNG values over the two experiments in the range of -2.5 to -0.1, values below the threshold of zero NNG required for a positive response. No cells were seen in repair at any dose of PROXEL Press Paste (BIT).
It may be noted that due to mortalities seen at the top dose tested in the 12-14 hour experiment, it was only possible to analyse UDS from two animals. In view of the clear negative results obtained in this assay, this was not considered to have prejudiced the validity of the study.
The data obtained in this study indicate that oral treatment of male rats dosed once with 540 or 1400 mg/kg PROXEL Press Paste did not result in increased UDS in hepatocytes isolated approximately 12-14 or 2-4 hours after dosing.

Conclusion
When treated orally once with PROXEL Press Paste at doses up to 1400 mg/kg, male rats showed no induction of UDS in hepatocytes isolated ex vivo approximately 12-14 or 2-4 hours after dosing. It is concluded that PROXEL Press Paste had no genotoxic activity detectable in this test system under the experimental conditions employed.
The study can be considered to be compatible with OECD 486. Although UDS analysis was not performed on three animals in the top dose group (due to a mortality) in the 12-14 hour test, it is not considered to affect the validity of the study since the results from the UDS analysis performed were clearly negative.

Executive summary:

A study was conducted to determine the in vivo genotoxic potential of the substance according to OECD Guideline 486. Groups of three male rats were treated with the test substance at 0, 560 and 1400 mg/kg bw by oral gavage. The positive controls used were 75 mg/kg bw 2 acetamidofluorene (2 -AAF) suspended in corn oil (12 -14 hour experiment) and 10 mg/kg bw dimethylnitrosamine (DMN) dissolved in purified water (2 -4 hour experiment). Approximately 12 -14 hours (experiment 1) or 2 -4 hours (experiment 2) after dosing, animals were killed and their livers were perfused with collagenase to provide a primary culture of hepatocytes and the cultures were treated with [3H] thymidine. Slides from the cultures were evaluated to determine the net grain count as a measure of the unscheduled DNA synthesis. Negative (vehicle) control animals gave group mean net grain count of  2.3 and 0 in experiments 1 and 2, respectively. These values did not exceed the upper limit of the historical negative control range. Group mean net grain count values were increased by 2 -AAP and DMN treatment. Overall, the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with the test substance up to 1400 mg/kg bw yielded net grain values less than zero, producing group mean values in the range of -2.5 to -0.1, values below the threshold required for a positive response. Under the study conditions, the substance was not considered to be genotoxic in the primary hepatocyte cultures in rat (Howe, 2001).

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

The Nipacide BIT used in this study had a reported organic purity of 99.8% of which 98.8% is BIT.
Batch A 88

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were 4-5 weeks old when received from the breeding unit. The animals were acclimatised for at least 4 days prior to treatment.

Route of administration:

oral: gavage

Vehicle:

Corn Oil

Duration of treatment / exposure:

1 oral (gavage) dose. Post exposure periods of 24, 48 and 72 hours

Post exposure period:

Post exposure periods of 24, 48 and 72 hours

Remarks:

Doses / Concentrations:
125, 250, 500, 1000, 2000 and 5000 mg/kg bw

No. of animals per sex per dose:

5-15

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive Control: Chlorambucil, administered once orally at a dosage of 30 mg/kg in aqueous 10% ethanol.

Tissues and cell types examined:

bone marrow

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

There was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of Nipacide BIT.

Conclusions:

Materials and methods
The effect of Nipacide BIT on chromosome structure in bone marrow cells was investigated following acute oral administration to mice according to OECD 474 (1983). Chromosome damage was measured indirectly by counting micronuclei.
A preliminary toxicity test was conducted using dosages of 125, 250, 500, 1000, 2000 and 5000 mg/kg. Adverse reactions to treatment were observed in all mice dosed at 2000 and 5000 mg/kg, two mice dosed at 1000 mg/kg and a single mouse dosed at 500 mg/kg. Two mice dosed at 5000 mg/kg and one mouse dosed at 2000 mg/kg were found dead or killed in extremis within 24 hours of dosing. Subsequently, in the main micronucleus test, male and female mice were given a single dose of Nipacide BIT at 300, 600 or 1200 mg/kg. In all cases Nipacide BIT was dosed orally, suspended in corn oil. Concurrent vehicle and positive control groups of mice were similarly dosed with corn oil or chlorambucil (30 mg/kg) respectively. Five males and five females from each group were killed 24 hours after treatment; further lots of five males and five females, given Nipacide BIT at 1200 mg/kg or the vehicle control, were killed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination.
At least 2000 erythrocytes per animal were then examined for the presence of micronuclei, using the light microscope. Calculated values of micronuclei per 1000 polychromatic erythrocytes were analysed statistically using the Mann-Whitney U test. The ratio of polychromatic:mature cells was also calculated for each group, as an indicator of gross toxicity.

Results and discussion
Adverse reactions to treatment were observed in all thirty mice dosed with Nipacide BIT at 1200 mg/kg, including hunched posture (30 mice), piloerection (28), underactivity (14), noisy respiration (7), partially closed eyes (5), prostrate posture (2), distended abdomen (1), abnormal gait (1) and skin pallor (1); four of these mice were found dead between 2 and 47 hours after dosing. Transient reactions to treatment were also observed in two mice dosed at 600 mg/kg. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any group treated with Nipacide BIT.
Frequencies of micronucleated polychromatic erythrocytes in animals killed 24, 48 or 72 hours after administration of Nipacide BIT were similar to those in concurrent vehicle controls. This lack of treatment related effect was apparent in both sexes, and was confirmed by statistical analysis. The sensitivity of the test was shown by statistically significant increases in the frequency of micronucleated polychromatic erythrocytes over control values in positive control group animals given chlorambucil at 30 mg/kg (p<0.01).
It is concluded that, under the conditions of test, there was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of Nipacide BIT.

Conclusion
There was no evidence of induced chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes of treated mice 24, 48 or 72 hours after oral administration of Nipacide BIT.

Executive summary:

A study was conducted to determine the in vivo genotoxic potential of the substance according to OECD Guideline 474. Male and female mice were given a single oral gavage of test substance at 300, 600 or 1200 mg/kg bw. Concurrent vehicle and positive control groups of mice were dosed with corn oil or chlorambucil (30 mg/kg bw) respectively. Five males and five females from each group were sacrificed 24 hours after treatment and five males and five females at 1200 mg/kg bw or the vehicle control were sacrificed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination. At least 2000 erythrocytes per animal were examined for the presence of micronuclei, using the light microscope. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any treated group. Frequencies of micronucleated polychromatic erythrocytes in all test groups were similar to those in concurrent vehicle controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Under the study conditions, the substance was not considered to be genotoxic in the micronucleus assay in mice (Edwards, 1995).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro:

A study was conducted to determine the in vitro genotoxic potential of the substance according to a method equivalent to OECD Guideline 471. The test substance was evaluated in the bacterial mutagenicity assay, using five strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA98 and TA100). Based on the cytotoxicity of the test substance in the absence and presence of metabolic activation (80 µg/plate and 200 µg/plate, respectively) in a range-finding study, the main assay was conducted at concentrations from 0.064 to 80 µg/plate without metabolic activation and from 0.32 to 200 µg/plate with metabolic activation. Each plate was prepared by the plate incorporation procedure. After incubation of the plates, the revertant colonies were counted using an automated colony counter (AMS 40-10 Image Analyser).  The test data were analysed for validity and for any reproducible dose related increase in revertant colonies.  Statistical analysis was performed using a one tailed Student’s t-test. The test substance induced significant cytotoxicity in all strains at 80 µg/plate in the absence of metabolic activation and 200 µg/plate in the presence of metabolic activation. The positive control data for each strain tested showed evidence of a mutagenic response which was dose related in the absence and presence of metabolic activation. Under the study conditions, the substance was not considered to be mutagenic in S. typhimurium strains in the presence and absence of metabolic activation (Callander, 1989).

[It should be noted that in a study performed by the same laboratory (Callander R.D; 1988; Central Toxicological Laboratory Report No. CTL/P/2056) using the same strains of S. typhimurium and dose ranges of 300 to 4.0 µg/plate, 250 to 0.8 µg/plate (both with metabolic activation) and 200 to 0.8 µg/plate (without metabolic activation), PROXEL Press Paste induced reproducible, dose-related, significant increases in the observed numbers of revertant colonies in strains TA1538 and TA98 in the presence of metabolic activation.  These effects were not observed in strains TA1535, TA1537 or TA100 in the presence of metabolic activation.  In the absence of metabolic activation, PROXEL Press Paste did not induce any significant reproducible increases in the observed numbers of revertant colonies in any of the five tester strains used.    

In another study (May K.; 1995; Pharmaco-LSR Ltd., Report No. 94/NLL051/0788) which tested the mutagenic effects of Nipacide BIT at dose ranges of 100 to 1.0 µg/plate (with and without metabolic activation) on strains TA98, TA100, TA1535 and TA1537, there was no evidence of mutagenic activity under the conditions of the test.  Trueman R.W, 1979 (Central Toxicological Laboratory Report No. CTL/R/494) tested strains TA1535, TA1537, TA1538, TA100 and TA98 at a dose range of 2500 to 0.32 µg/plate (with and without metabolic activation) and there was no mutagenic effect observed in the presence or absence of metabolic activation.

When the data from May (1995) and Trueman (1979) are considered together with the Callander (1989) data (presented in this summary) it can be concluded that the presence of 1,2-Benzisothiazolin-3(2H)-one is unlikely to evoke mutagenic activity in this type of assay.  The positive result observed in Callander (1988) was not observed in three other studies performed under similar conditions.]

A study was conducted to determine the genotoxic potential of the substance in mammalian cells according to OECD Guideline 476. The test substance was assayed for mutation at the hprt locus (6-thioguanine resistance) in mouse lymphoma cells. The study consisted of cytotoxicity range-finding experiments followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254 induced rat liver post-mitochondrial fraction (S-9). The initial cytotoxicity range-finding experiment with and without metabolic activation was performed in the concentration range 46.88 to 1512 µg/mL.  Since complete toxicity was observed at all concentrations, a second range-finding experiment was performed with and without metabolic activation in the range 0.3906 to 50 µg/mL. On the basis of the results from this second range-finding experiment, Experiment 1 of the main test was performed in the concentration range 0.1 to 3.2 µg/mL in the absence of metabolic activation and in the concentration range 0.2 to 6.4 µg/mL in the presence of metabolic activation.  The concentration range was extended in Experiment 2 of the main test to 0.2 to 6.4 µg/mL in the absence of metabolic activation and to 0.4 to 12.8 µg/mL in the presence of metabolic activation. 4 Nitroquinoline 1 -oxide (in the absence of metabolic activation) or Benzo(a)pyrene (in the presence of metabolic activation) were used as the positive controls in this study. Mutant frequency was assessed for statistical significance. In experiment 1, the highest dose selected in the presence of S-9 (6.4 µg/mL) could not be analysed due to excessive contamination observed on the mutant plates and the second highest dose tested (4.8 µg/mL) was considered too toxic for selection to determine viability and 6 -thioguanine (6TG) resistance. The highest dose analysed in both the absence and presence of S-9 was therefore 3.2 µg/mL, with relative survival being 61% and 44%, respectively. No dose of ideal toxicity (10-20% relative survival) was achieved in the absence or presence of S-9. However, adequate dose-related toxicity was demonstrated in the absence and presence of S-9 in Experiment 2, therefore this was not considered to have affected the integrity of the study in any way. In experiment 2, the highest doses analysed were 4.8 µg/mL in the absence of S-9 and 6.4 µg/mL in the presence of S 9, with relative survival being 9% and 10%, respectively. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive controls. The study was therefore considered valid. No statistically significant increases in mutant frequency were observed following treatment at any of the non-toxic doses analysed. Under the study conditions, the substance was not considered to be mutagenic in mouse lymphoma cells (Lloyd, 2003).

A study was conducted to determine the in vitro genotoxic potential of the substance according to OECD Guideline 473. The effects of the test substance on chromosomal structure was investigated in cultured human lymphocytes with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix).  In the absence of metabolic activation, cells were exposed continuously for 21 and/or 45 hours; with metabolic activation, exposure was limited to three hours and cells were harvested 18 or 42 hours later (again resulting in sampling times of 21 or 45 hours). Treatments were established by the addition of test solutions in dimethyl sulphoxide at 1 to 32  µg/mL (experiment 1) or at  24 to 40 µg/mL (repeat experiment) and at 2 to 16 µg/mL (experiment 2) to 48-hour cultures of whole, human blood. Cell division was arrested by the addition colcemid three hours before the cells were harvested and slides were then prepared for microscopic analysis. Mitotic indices were calculated for each culture based on the number of metaphases observed per 1000 cells scored. Chromosome aberrations were scored by examination of 100 metaphases per culture and the frequencies of cells with one or more aberrations were calculated both including and excluding gap-type aberrations. In the absence of metabolic activation significant cytotoxicity was observed at 32 µg/mL and in the presence of metabolic activation, cytotoxicity was observed at 24 or 32 µg/mL. In the absence of metabolic activation, the highest concentrations selected for chromosomal analysis produced statistically significant increases in the frequency of chromosomal aberrations at the 21 hour sampling time. The increases exceeded the historical solvent control range at the testing laboratory and were reproducible. The positive and negative controls were considered valid. Under the study conditions, the substance was considered to be genotoxic in the chromosomal aberration assay with cultured human lymphocytes (Edwards, 1995).

In vivo:

A study was conducted to determine the in vivo genotoxic potential of the substance according to OECD Guideline 486. Groups of three male rats were treated with the test substance at 0, 560 and 1400 mg/kg bw by oral gavage. The positive controls used were 75 mg/kg bw 2 acetamidofluorene (2 -AAF) suspended in corn oil (12 -14 hour experiment) and 10 mg/kg bw dimethylnitrosamine (DMN) dissolved in purified water (2 -4 hour experiment). Approximately 12 -14 hours (experiment 1) or 2 -4 hours (experiment 2) after dosing, animals were killed and their livers were perfused with collagenase to provide a primary culture of hepatocytes and the cultures were treated with [3H] thymidine. Slides from the cultures were evaluated to determine the net grain count as a measure of the unscheduled DNA synthesis. Negative (vehicle) control animals gave group mean net grain count of  2.3 and 0 in experiments 1 and 2, respectively. These values did not exceed the upper limit of the historical negative control range. Group mean net grain count values were increased by 2 -AAP and DMN treatment. Overall, the system was shown to be sensitive to two known DNA damaging agents requiring metabolism for their action. The assay was therefore accepted as valid. Treatment with the test substance up to 1400 mg/kg bw yielded net grain values less than zero, producing group mean values in the range of -2.5 to -0.1, values below the threshold required for a positive response. Under the study conditions, the substance was not considered to be genotoxic in the primary hepatocyte cultures in rat (Howe, 2001).

A study was conducted to determine the in vivo genotoxic potential of the substance according to OECD Guideline 474. Male and female mice were given a single oral gavage of test substance at 300, 600 or 1200 mg/kg bw. Concurrent vehicle and positive control groups of mice were dosed with corn oil or chlorambucil (30 mg/kg bw) respectively. Five males and five females from each group were sacrificed 24 hours after treatment and five males and five females at 1200 mg/kg bw or the vehicle control were sacrificed 48 and 72 hours after treatment. Bone marrow smears on glass slides were made from each animal. These slides were then stained and prepared for examination. At least 2000 erythrocytes per animal were examined for the presence of micronuclei, using the light microscope. No real indication of bone marrow toxicity, as evidenced by depression of bone marrow proliferation, was noted in any treated group. Frequencies of micronucleated polychromatic erythrocytes in all test groups were similar to those in concurrent vehicle controls. This lack of treatment-related effect was apparent in both sexes, and was confirmed by statistical analysis. Under the study conditions, the substance was not considered to be genotoxic in the micronucleus assay in mice (Edwards, 1995).

Justification for classification or non-classification

Based on the in vitro and in vivo genotoxicity studies with the substance, no classification for mutagenicity is warranted according to EU CLP (1272/2008) criteria.