1,3,5-tris(2,3-dibromopropyl)-1,3,5-triazine-2,4,6(1H,3H,5H)-trione

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for mutagenicity and genotoxicity according to the following: 

• In vitro gene mutation in bacteria, according to OECD Guideline 471 (Bacterial Reverse Mutation Assay);


• In vitro cytogenicity in mammalian cells, according to a test protocol equivalent to OECD Guideline 473 (In vitro mammalian chromosome aberration test);


• In vitro gene mutation study in mammalian cells, according to a test protocol equivalent to OECD Guideline 476 (In vitro mammalian cells gene mutation test);


• In vitro transformation studies in mammalian cells (CTA);

All studies except the Bacterial Reverse Mutation Assay were conducted prior to the first approved version of the corresponding OECD guidelines, thus these sources of information are addressed in a weight of evidence approach or as supporting information. 

 

The following conclusions were obtained:

• the results of the Bacterial Reverse Mutation Assay indicate that the test item was positive with tester strains TA100 and TA1535 both with and without S9;


• the test item did not show evidence of mutagenic activity to cultured human lymphocytes in vitro at the dose levels tested, both with and without metabolic activation in any of the tests performed;


• the negative results of the mammalian cells gene mutation study reported lend support to the conclusion that the test item is unlikely to represent a mutagenic hazard to man;


• the test item did not show transforming ability and by the criteria of the CTAs performed and cannot be considered a potential carcinogen.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

human lymphocites

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

June 1982

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Significant deficiencies from current Guideline: +S9 trial treatment time 2 hrs (guideline calls for 3-6); top dose (highest doseable concentration) did not show toxicity; questionable adequate number of cells examined (100 metaphases as opposed to 300).

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test Material: Interstab FR 930
Also used as comparison with another substance: Y 00928/008/001

Species / strain / cell type:

lymphocytes: humans

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

For metaphase analysis without metabolic activation, the test agent FR 930 was added to the lymphocyte cultures at final concentrations of 100, 500 and 1000 ug/ml after 48 hours incubation.

For metaphase analysis with metabolic activation, lymphocytes were harvested after 48 hours of incubation and resuspended in tissue culture medium containing S-9 mix. FR 930 was added to the cultures at final concentrations of 100, 500 and 1000 ug/ml.

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Positive control compounds: mitomycin C (without metabolic activation) and cyclophosphamide (with metabolic activation), in order to confirm the sensitivity of the test system to the effects, respectively, of a direct acting and indirect acting mutagen.

Evaluation criteria:

Metaphase analysis for number and type of chromosome aberrations and cell counts referring to Fisher Method

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Refer to Table 3 of full study report for details.

Positive controls validity:

valid

Additional information on results:

FIRST EXPERIMENT
Preliminary toxicity test on Interstab FR 930:
From the results obtained by the viable cell counts, it was indicated that a top dosage of 1000 ug/ml could be used for the metaphase analysis study, this being the maximum possible dosable concentration.

Metaphase analysis of Interstab FR 930:
The number of chromatid breaks and acentric fragments recorded for all concentrations of FR 930 in the absence of metabolic activation was comparable with the solvent control value. The number of aberratiors (excluding gaps) recorded for FR 930 was not significantly greater than the solvent control value using the Fisher's test (P> 0.05).
The number of chromatid breaks and acentric fragments recorded for all concentrations of FR 930 in the presence of metabolic activation was comparable with the solvent control value. The number of aberrations (excluding gaps) recorded for FR 930 was not significantly greater than the solvent control value using the Fisher's test (P>0.05).
Dimethylsulphoxide, the solvent control, produced only a very low number of chromatid breaks and acentric fragments both in the presence and absence of metabolic activation.
In the absence of metabolic activation mitomycin C, at a final concentration of 0.5 ug/ml, produced an increased number of chromatid breaks and acentric fragments compared with the solvent control. One chromatid exchange was also observed. The number of chromatid breaks and chromatid exchanges was significantly greater than the solvent control values, using the Fisher's test (P<0.05).
In the presence of metabolic activation, cyclophosphamide at a final concentration of 40 ug/ml produced an increased number of chromatid breaks and acentric fragments compared with the solvent control. Three chromatid exchange figures were also observed. The number of chromatid breaks and chromatid exchanges was significantly greater than the solvent control values, using the Fisher's test (P<0.01).

SECOND EXPERIMENT
Preliminary toxicity test on Y 00928/008/001:
From the results obtained by the viable cell counts, it was indicated that a top dosage of 20 ug/ml should be used for Y 00928/008/001 in the metaphase analysis both in the presence and absence of metabolic activation. The results of the preliminary toxicity test on Interstab FR930 carried out for the first experiment, indicated that a top dosage of 1000 ug/ml should be used for the metaphase analysis both in the presence and absence of metabolic activation.

Metaphase analysis of Interstab FR 930 and Y 00928/008/001:
The percentage of cells showing aberrations for all concentrations of FR 930 and Y 00928/008/001 in the absence of metabolic activation was comparable with the solvent control value.
The percentage of cells showing chromosome aberrations for all concentrations of Y 00928/008/001 in the presence of metabolic activation was marginally higher than the solvent control value. None of these increases was found to be significant (P>5%) using Fisher's test.
The percentage of aberrant metaphases recorded for all concentrations of FR 930 in the presence of metabolic activation was comparable with the solvent control value. Dimethylsulphoxide, the solvent control, produced only a low number of chromatid breaks and acentric fragments both in the presence and absence of metabolic activation.
In the absence of metabolic activation, mitomycin C, at a final concentration of 0.5 ug/ml, produced a large number of chromatid breaks and acentric fragments compared with the solvent control. One chromatid exchange was also observed. It was not possible to score 100 cells at this concentration, as few metaphase spreads were observed, due to the toxicity of the compound. The increase in the number of chromatid breaks and chromatid exchanges was significant (P<0.1%) using Fisher's test.
In the presence of metabolic activation, cyclophosphamide at a final concentration of 50 ug/ml produced an increased number of chromatid breaks and acentric fragments compared with the solvent control. Eight chromatid exchange figures were also observed. The increase in the number of chromatid breaks and chromatid exchanges was significant (P<0.1%) using Fisher's test.

DISCUSSION
The number of aberrant metaphases observed at all three dose levels of FR 930, both in the presence and absence of metabolic activation, in the second experiment were similar to the respective concurrent solvent control group values. These results confirmed the negative findings of the first experiment.
The number of aberrant metaphases observed at all three dose levels of Y 00928/008/001 in the absence of metabolic activation were comparable with the concurrent solvent control group value. However Y 00928/008/001 gave slightly aberrant metaphase counts at all three dose levels in the presence of metabolic activation. The increase for each dose level wes not significantly different from the control value using Fisher's test but if the numbers of aberrant metaphases for all three dose levels were pooled a significant difference from the solvent control value ws demonstrated (P<0.05). The increases are suggestive of slight mutagenic activity.
Y 00928/008/001 has shown evidence of mutagenic activity in bacteria using the Ames test and Ames test on the urine of tested animals. The compound has produced increases in sister chromatid exchanges using LC5178Y mouse lymphoma cells and chinese hamsters V79 cells as well as producing malignant transformation in the BALB/3T3 cell system. The compound has also been shown to induce sex-linked recessive lethal mutations in the fruit fly Drosophila melanogaster and to increase the incidence of chromosome aberrations in L5178Y cells 1 and V79 cells when these cells are analysed at the metaphase stage of cell division in vitro. However, the compound has given negative results for chromosome aberrations in rat bone marrow cells in vivo and human lymphoid cells JEFF and Birkitt B 35M in vitro. From these results it is clear that the compound does not give a clear positive response in all chromosome aberration test systems.
The lack of any toxicity caused by Y 0928/008/001 at the top dose level chosen for the second experiment is probably due to a lack of reliability in the method of assessing viability used in the study. However, if the mitotic indices recorded for both experiments are compared with the cell viabilities, it can be seen that the two sets of values do not precisely correlate. This is probably because cell viability measures the immediate response to the dose (and does not recognise any repair capacity), whereas the mitotic index may be a measure of the suppresion of cell cycle activity following treatment and is not necessarily a good indication of compound induced toxicity. Assays involving the culture of human lymphocytes do not permit the use of more conventional estimations of cytotoxicity such as measurement of plating efficiency or colony forming ability.

It should also be noted that Interstab FR 930 was added to the cultures at the maximum possible concentration in the main study and no increases in aberrant metaphases were detected. However, Y 00928/008/001 which could have been dosed at higher concentrations nevertheless showed some indication of mutagenic activity, suggesting that if higher dose levels had been used, a clearer distinction between the mutagenic potential of the 2 test agents would have been possible.

Conclusions:

FR 930 was tested in vitro to determine whether it causes chromosomal aberrations in cultured human lymphocytes.
One month later, the experiment on FR 930 was repeated togheter with another test compound Y 00928/008/001.
A preliminary toxicity test for the first experiment, was carried out to assess the effect of FR930 on the viability of cultured human lymphocytes. The result showed that a top concentration of 1000 ug/ml, which was the highest dosable concentration, would not cause too large a reduction in the viability of the cells.
Human lymphocytes were grown in tissue culture medium at 37°C, and mitotic cell division was simulated by addition of phytohaemagglutinin.
For the metaphase analysis without metabolic activation in the first experiment, the test agent, FR 930 was added to the lymphocyte cultures at final concentrations of 100, 500 and 1000 ug/ml after 48 hours’ incubation. The cultures were incubated for a further 24 hours. Dimethylsulphoxide was used as the solvent control, and mitomycin C at a final concentration of 0.5 ug/ml was used as the positive control.
For the metaphase analysis with metabolic activation in the first experiment, lymphocytes were harvested after 48 hours' incubation and resuspended in tissue culture medium containing S-9 mix. FR930 was added to the cultures at final concentrations of 100, 500 and 1000 ug/ml. The cultures were incubated with the test compound for two hours and the cells were then harvested, washed and resuspended in tissue culture medium. The cultures were incubated for a further 22 hours. The positive control agent used was cyclophosphamide at a final concentration of 40 ug/ml and dimethylsulphoxide was used as the solvent control.
The lymphocytes were arrested in the metaphase stage of cell division, fixed stained and examined microscopically for evidence of chromosomal damage.
For the second experiment, a preliminary toxicity test was carried out to assess the effect of Y 00928/008/001 on the viability of cultured human lymphocytes. The results showed that a top concentration of 20 ug/ml of Y 00928/008/001 would not cause too large a reduction in the viability of the cells.
For the metaphase analysis without metabolic activation in the second experiment, FR 930 was added at final concentration of 100, 500 and 1000 ug/ml and Y 00928/008/001 was added to the cultures at final concentrations of 2, 10 and 20 ug/ml after 48 hours incubation. The cultures were incubated for a further 24 hours. Dimethylsulphoxide was used as the solvent control, and mitomycin C at a final concentration of 0.5 ug/ml was used as the positive control.
For the metaphase analysis with metabolic activation in the second experiment, FR 930 was added at final concentration of 100, 500 and 1000 ug/ml and Y 00928/008/001 at final concentrations of 2, 10 and 20 ug/ml. Cyclophosphamide at a final concentration of 50 ug/ml was used as the positive control and dimethylsuphoxide used as the solvent control.
The results of the first experiment, showed that cultures treated with FR 930, did not show any significant increase in the amount of detectable chromosomal damage when compared with the concurrent solvent control treated cultures, either in the presence or absence of metabolic activation by S-9 mix.
The result of the second experiment, showed that cultured cells treated with FR 930 both in the presence and absence of metabolic activation and those treated with Y 00928/008/001 in the absence of metabolic activation did not show any increase in the amount of detectable chromosomal damage when compared with the concurrent solvent control treated cultures. However, Y 00928/008/001 produced marginal increases at all dose levels in the proportion of aberrant metaphases when treated in the presence of metabolic activation but these increases were not significant in Fisher’s test.
The results obtained with the positive control compounds, mitomycin C and cyclophosphamide, in both experiments, confirmed the sensitivity of the test system to the effects, respectively, of a direct acting and indirect acting mutagen.

From the result obtained in both experiment, it was concluded that FR 930 did not show evidence of mutagenic activity. From the results obtained in the second experiment, Y 00928/008/001 showed slight evidence of mutagenic activity when tested with cultured human lymphocytes in vitro at the dose levels used and in presence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

human lymphocites

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March 1982

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

yes

Remarks:

Significant deficiencies from current Guideline: +S9 trial treatment time 2 hrs (guideline calls for 3-6); top dose (highest doseable concentration) did not show toxicity; questionable adequate number of cells examined (100 metaphases as opposed to 300)

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Test material: Interstab FR 930

Species / strain / cell type:

lymphocytes: humans

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

For metaphase analysis without metabolic activation, the test agent FR 930 was added to the lymphocyte cultures at final concentrations of 100, 500 and 1000 ug/ml after 48 hours incubation.

For metaphase analysis with metabolic activation, lymphocytes were harvested after 48 hours of incubation and resuspended in tissue culture medium containing S-9 mix. FR 930 was added to the cultures at final concentrations of 100, 500 and 1000 ug/ml.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Positive control compounds: mitomycin C (without metabolic activation) and cyclophosphamide (with metabolic activation), in order to confirm the sensitivity of the test system to the effects, respectively, of a direct acting and indirect acting mutagen.

Evaluation criteria:

Metaphase analysis for number and type of chromosome aberrations and cell counts reffering to Chi-squared test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Refer to Table 3 of full study report for details.

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST:
From the results obtained by the viable cell counts, it was indicated that a top dosage of 1000 ug/mI should be used for the metaphase analysis.

METAPHASE ANALYSIS:
The number of chromatid breaks and accentric fragments recorded for all concentrations of FR 930 in the absence of metabolic activation was comparable with the solvent control value. The number of aberrations (excluding gaps) recorded for FR 930 was not significantly greater than the solvent control value using the chi-square exact test (P>0.05) .
The nunber of chromatid breaks and acentric fragments recorded for all concentrations of FR 930 in the presence of metabolic activation was comparable with the solvent control value. The number of aberrations (excluding gaps) recorded for FR 930 was not significantly greater than the solvent control value using the chi-square exact test (P> 0.05) .
Dimethyl sulphoxide, the solvent control, produced only a very low number of chromatid breaks and acentric fragments both in the presence and absence of metabolic activation.
In the absence of metabolic activation mitomycin C, at a final concentration of 0.5 ug/ml, produced an increased number of chromatid breaks and acentric fragments compared with the solvent control. One chromatid exchange was also observed. The number of chromatid breaks and chromatid exchanges was significantly greater than the solvent control values, using the chi-square exact test (P<0.05) .
In the presence of metabolic activation, cyclophosphamide at a final concentration of 40 ug/ml produced an increased number of chromatid breaks and acentric fragments compared with the solvent control. Three chromatid exchange figures were also observed. The number of chromatid breaks and chromatid exchanges was significantly greater than the solvent control values, using the chi-square exact test (P < 0.01).

Conclusions:

FR 930 was tested in vitro to determine whether it causes chromosomal aberrations in cultured human lymphocytes.
A preliminary toxicity test was carried out to assess the effect of FR930 on the viability of cultured human lymphocytes. The results showed that a top concentration of 1000 µg/ml would not cause too large a reduction in the viability of the cells.
Human lymphocytes were grown in tissue culture medium at 37°C, and mitotic cell division was stimulated by the addition of phytohaemagglutinin.
For metaphase analysis without metabolic activation, the test agent FR 930 was added to the lymphocyte culture at final concentration of 100, 500 and 1000 µg/ml after 48 hours incubation. The cultures were incubated for a further 24 hours. Dimethyl sulphoxide was used as the solvent control, and mitomycin C at a final concentration of 0.5 µg/ml was used as the positive control.
For metaphase analysis with metabolic activation, lymphocytes were harvested after 48 hours’ incubation and resuspended in tissue culture medium containing S-9 mix. FR 930 was added to the cultures at final concentrations of 100, 500, 1000 µg/ml. The cultures were incubated with the test compound for two hours and the cells were then harvested, washed and resuspended in tissue culture medium. The cultures were incubated for a further 22 hours. The positive control agent used was cyclophosphamide at a final concentration of 40 µg/ml and Dimethyl sulphoxide was used as the solvent control.
The lymphocytes were arrested in the metaphase stage of cell division, fixed, stained and examined microscopically for evidence of chromosome damage.
Cultures treated with FR 930 did not show any significant increase in the amount of detectable chromosome damage when compared with the concurrent solvent control-treated cultures, either in the presence or absence of metabolic activation by S-9 mix.
The results obtained with the positive control compounds, mitomycin C and cyclophosphamide, confirmed the sensitivity of the test system to the effects, respectively, of a direct acting and indirect acting mutagen.

From the result obtained, it was concluded that FR 930 did not show evidence of mutagenic activity to cultured human lymphocytes in vitro at the dose levels tested, both with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 1982

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

yes

Remarks:

Significant deficiencies from current Guideline, e.g. treatment time 2 hours (instead of 3-6); questionable use of adequate number of cells used; no TA identification.

GLP compliance:

yes

Remarks:

GLP compliance statement included in the Test Report

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

Test Material: Interstab FR 930

Target gene:

HGPRT - Hypoxanthine-Guanine PhosphoRibosylTransferase

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Chinese hamster ovary cells designated CHO-KI-BH4 derived from a clone isolated (O 'Neill , Brimer Machanoff, Hirsch & Hsie , 1977) from a line originally isolated by Kao & Puck (1967) were obtained in 1978 from Dr O'Neill (Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA) .They are stored in liquid nitrogen.

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

The studies employed 500, 250, 125 and 62 ug/ml of FR930 as well as negative and positive controls.

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

CELL LINE
Chinese hamster ovary cells designated CHO-KI-BH4 derived from a clone isolated (O 'Neill , Brimer Machanoff, Hirsch & Hsie , 1977) from a line originally isolated by Kao & Puck (1967) were obtained in 1978 from Dr O'Neill (Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA). They are stored in liquid nitrogen.

RECOVERY OF CELLS FOR STUDY
Cells were recovered from liquid nitrogen and grown for 10 days before the start of the stu

CULTURE MEDIUM
Basic medium:
A single batch of basic medium was prepared the start of the study. This consisted of nutrient mixture F—12 (Ham) single strength, buffered with sodium bicarbonate and containing
L—glutamine (146 mg/ lit re) . It was supple— men ted as follows :
solution of penicillin (10,000 IU/m1) and streptomycin (10,000 y g/ml): 10 ml/litre medium
solution of L-glutamine (200 mM): 10 ml/litre medium
foe tal calf serum: 50 ml/litre medium

Selective medium:
A single batch of hypoxanthine—free medium was prepared at the start of the mutation studies . Hypoxanthine—free nutrient mixture F—12 (Ham) was supplemented with antibiotics, glutamine and foe tal calf serum.
Thioguanine was added to the mutant selection plates after seeding with cells.

Evaluation criteria:

The results of the mutation test (mutant frequencies) were interpreted according to the criteria laid down in Protocol I 3.422.41, paragraph 7.3., as follows:

"The test article will be considered mutagenic if:
1. The negative control data are within the normal range of the spontaneous mutation frequency for the HGPRT locus.
2. The positive control data are comparable with those obtained previously with the positive control article.
3. A mutation frequency at least 2.5 times greater than the frequency shown by the concurrent negative control is observed at one or more concentration of the test article.
4. A dose response relationship is observed over some of the range of test concentrations."

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

PRELIMINARY TOXICITY TEST
No evidence of cytotoxicity was observed with concentrations of Interstab FR 930 up to and including 200 ug/ml when S-9 mix was not used. It was concluded that the maximum concentration which could be used in the mutation test without S-9 was in excess of 200 ug/ml. Thus 500 ug/ml the highest concentration possible due to limited solubility in DMSO was therefore chosen.
In the presence of S-9 mix, and 200 or 20 mg/l none of the cells survived. There was no toxicity at 2 ug/ml and it was assumed that some effect would be apparent at concentration between 2 and 20 ug/ml. Accordingly, 10 ug/ml was chosen as the highest concentration for the first study of mutation.

1ST STUDY MUTATION
In the absence of S-9 mix, none of the concentrations of test article showed any evidence of cytotoxicity and according to the evaluation criteria was judged negative for mutation induction. The positive control, EMS showed a clear mutagenic effect.
In the presence of S-9 mix, there was no evidence of cytotoxicity up to 10 ug/ml Interstab FR 930. The culture treated with 5 ug/ml was through infection and could not be evaluated for mutant frequency. The mutant frequencies observed were not greater than 2.5 times the control. A positive response, according to the evaluation criteria cited, was seen with the positive control (50 ug/ml) CPA.

SUPPLEMENTARY STUDY OF MUTATION
Treatment with concentrations of test article up to 500 ug/ml in the presence of S-9 mix had no effect on cell survival. In this study, the back ground frequency of mutants was much higher than in the first but compared with the lower control counts seen in the other phases of the study, there is no evidence of a dose related increase in mutant frequency over the range of concentration of test article.

2ND STUDY OF MUTATION
In the second study of mutation the same range of test article concentrations was used with and without S-9 mix. The highest concentration (500 ug/ml), gave a mutant frequency in the absence of S-9 mix which was just over 3 times the control value. The mutant frequencies with all other treatments were similar to or lower than the negative control.

DISCUSSION
Experience in this laboratory has shown that the background mutant frequency observed in the CHO/HGPRT mutation test is in the range 0 to 25 mutants per million surviving cells. An historical mean of all studies is 6.7 (standard deviation 6.3). with added S-9 mix the mean value is 9.8 (standard deviation 9.9). This variation is observed not only between separate experiemnts but also between control cultures in the same experiment. For a mutation test to be judged positive, it is essential that induced mutation frequencies should either be above about 30 or should show a good dose response relationship. Only a combination of both criteria is really convincing. Taking the less stringent criterion of an increase above the study control value by a factor of 2.5, only one value (at 500 ug/ml without s-9 mix) could have been considered positive. This response was not repeatable and, in the absence of any evidence for a dose response relationship, is not considered to be a significant observation.
It should be noted that the range of concentrations of the test article which could be tested was limited by the solubility of the compound in DMSO, the solvent of choice in the study. The solubility of the test article in culture medium was considerably less than in DMSO as shown by the formation of a precipitate when DMSO solutions were added to medium. The results of in vitro tests on compounds which are essentially insoluble in water must be regarded with some caution.
Despite the limitations of the tests of the type employed in this study, the negative results of the mutation tests reported here for mammalian cells lend support to the conclusion that FR 930 is unlikely to represent a mutagenic hazard to man.

Conclusions:

The potential of FR 930 to induce mutations in mammalian cells has been assessed using a modification of the CHO/HGPRT mutation test developed by O’Neill, Brimer, Machanoff, Hirsch and Hsie (1977). This test uses cultured Chinese hamster ovary cells (CHO cells) which after chemical treatment are maintained in a growing state for an extended period (the expression time). After this, mutant cells lacking hypoxantine-guanine phosphoribosyltranferase (HGPRT) are selected from the treated population by growth in medium containing thioguanine. An increase in the frequency of HGPRT-deficient cells in a treated population indicates that the treatment is mutagenic to CHO cells.
In this study, a 2-hr treatment period has been used followed by a 7-day expression time with mutant selection in 2 microg/ml thioguanine.
The maximum concentration of FR 930 which could be incorporated in culture medium was found to be 500 microg/ml. two separate mutation tests were carried out without the use of a metabolic activation system. These studies both employed 500, 250, 125 and 62 microg/ml FR930 as well as negative and positive controls. A week positive effect with 500 microg/ml was observed in one study but not in the other. The other concentrations of the test article were negative.
The effect of FR 930 was also tested with the addition, during treatment, of a metabolic activation mixture (S-9 mix) containing post-mitochondrial supernatant from Aroclor-induced rat liver. The first study carried out with S-9 mix was unsatisfactory and was repeated as a supplementary study. No evidence of mutagenicity was observed in this study at concentration ranging in 2-fold serial dilutions from 500 microg/ml down to 2 microg/ml of FR 930. A second study employing 500, 250, 125 and 62 microg/ml was also negative.
The results of the total study are therefore considered uniformly negative. Thus, under the condition of the test, FR930 appears to be without any mutagenic effect on CHO cells.
Tests of the type used in this study indicate the potential of the test article to induce mutations in the mammalian cells in vitro.

The conclusions which can be drawn from the results in terms of genetic hazard to intact organisms and particularly man are limited by the rather artificial conditions of the test system. Nevertheless, the negative results of the study reported here lend support to the conclusion that FR930 is unlikely to represent a mutagenic hazard to man.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 2008 - February 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Test material: FR 930

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

FR-930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 10007 and 5000 microg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and 5000 microg/plate without S9.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

other: ICR-191

Remarks:

without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoantracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

Test System Rationale:
The bacterial reverse mutation assay has been shown to be a sensitive, rapid and accurate indicator of the mutagenic activity of many materials including a wide range of chemical classes. By using several different tester strains, both base pair substitution and frameshift mutations can be detected. Salmonella and E. coli strains used in this assay are histidine and tryptophan auxotrophs, respectively, by virtue of conditionally lethal mutations in the appropriate operons. When these histidine (his-) or tryptophan (trp-) dependent cells are exposed to the test article and grown under selective conditions (minimal media with a trace amount of histidine or tryptophan), only those cells which revert to histidine (his-) or tryptophan (trp+) independence are able to form colonies. Trace amounts of histidine or tryptophan added to the media allow all the plated bacteria to undergo a few cell divisions, which is essential for mutagenesis to be fully expressed. his+ or trp+ revertants are readily discernable as colonies against the limited background growth of his- or trp- cells.

Tester Strains:
The tester strains used were the Salmonella histidine auxotrophs TA98, TA100, TA1535, and TA1537 (Ames et al. 1975) and the E. coli tryptophan auxotroph WP2uvrA (Green and Muriel 1976). Specific genotypes of the strains are shown in the full study report.
In addition to a mutation in either the histidine or tryptophan operons, the tester strains contain additional mutations that enhance their sensitivity to some mutagenic compounds. Mutation of the uvrA gene (E. coli) or the uvrB gene (Salmonella) results in a deficient DNA excision repair system that greatly enhances the sensitivity of these strains to some mutagens. Since the uvrB deletion extends through the bio gene, Salmonella tester strains containing this deletion also require the vitamin biotin for growth.
Salmonella tester strains also contain the rfa wall mutation, which results in the loss of one of the enzymes responsible for the synthesis of part of the lipopolysaccharide (LPS) barrier that forms the surface of the bacterial cell wall. The resulting cell wall deficiency increases permeability to certain classes of chemicals, such as those containing large ring systems (i.e., benzo[a]pyrene), that otherwise would be excluded by a normal cell wall.
Tester strains TA98 and TA100 also contain the pKM101 plasmid, which further increases the sensitivity of these strains to some mutagens. The suggested mechanism for this increased sensitivity is modification of an existing bacterial DNA repair polymerase complex involved with the mismatch-repair process.
Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. In contrast, tester strains TA100, TA1535, and WP2uvrA are reverted from auxofrophy to prototrophy by base substitution mutagens.

Source of Tester Strains:
Salmonella tester strains were received from Dr. Bruce Ames, Department of Biochemistry, University of California (Berkeley, CA).
E. coli tester strain WP2uvrA was received from the National Collection of Industrial Bacteria, Torrey Research Station, Scotland (United Kingdom).

Rationale for test conditions:

Cytotoxicity of the test article observed in tester stain TA100 is generally representative of that observed in the other Salmonella tester strains. Because of TA100's comparatively high spontaneous revertant frequency (revertant colonies/plate), gradations of cytotoxicity can be readily discerned from routine experimental variation. E. coli tester strain WP2uvrA does not have the rfa wall mutation possessed by the Salmonella strains; therefore a different range of cytotoxicity may be observed. Also, cytotoxicity of a test article in the presence of S9 may vary greatly from that observed in its absence, requiring that different dose ranges be evaluated in the mutagenicity assay with and without S9.

Evaluation criteria:

Criteria for a Positive Response
A test article is considered to have produced a positive response if it induces a dosedependent increase in revertant frequency that is 22.0-fold vehicle control values for tester strains TA98, TAI 00, and WP2uvrA, or 23.0-fold vehicle control values for tester strains TAl 535 and •TA1537. In addition, any response should be reproducible.

Criteriafor a Negative Response
A test article is considered to have produced a negative response if no dose-dependent,
22.0-fold or 23.0-fold increases are observed in tester strains TA98, TAI 00, and WP2uvrA, or TAI 535 and TA1537, respectively.

Criteria for an Equivocal Response
Even after repeated trials, a test article may produce results that are neither clearly positive nor clearly negative (e.g., responses that do not meet the dose-dependency or fold increase requirements but are reproducible). In those rare instances, the test article may be considered to have produced an equivocal response.

Other criteria also may be used in reaching a conclusion about the study results (e.g., comparison to historical control values, biological significance, etc.). In such cases, the Study Director will use sound scientific judgrnent and clearly report and describe any such considerations.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

Test Article Handling:
The test article was found to form an opaque, white, heterogenous, non-viscous suspension in deionized water at a concentration of approximately 100 mg/mL. The test article was found to fom a transparent, colorless, non-viscous solution in DMSO at a concentration of approximately 100 mg/mL, which was the most concentrated dose formulation prepared for treatment; it remained freely soluble at all succeeding lower dilutions prepared for the assay.

Dose Analyses:
Determination of stability, homogeneity and concentration of the dosing preparations was not conducted.

Dose Range-finding Assay:
FR-930 was evaluated in the dose range-finding assay in tester strains TA100 and WP2uvrA. Ten doses of test article, from 6.67 to 5000 µg/plate, were evaluated with and without S9 (one plate per dose; Trial 29938-A1). No cytotoxicity was observed with either tester strain in the presence or absence of S9 as evidenced by no dose-related decreases in revertant frequency. Normal bacterial background lawns were observed up to 667 µg/plate with both tester strains in the absence of S9, and with TA100 in the presence of S9. Normal bacterial backgound lawns were observed up to 1000 µg/plate with WP2uvrA in the presence of S9. Test article precipitate was observed on the plates with both tester strains at doses ≥1000 µg/plate with and without S9. The test article precipitate obscured the bacterial backgound lawns at ≥ 1000 µg/plate (≥ 3330 µg/plate for WP2uvrA with S9). Dose-related increases in revertant frequency were observed with TA100 in the presence and absence of S9.

Mutagenicity Assay:
Based upon the results of the dose range-finding assay, FR-930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and 5000 µg/plate without S9 (Trial 29938-B1). All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester strains evaluated with and without S9 except at the highest 1-2 doses where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98, TA1537, and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA100 at doses of ≥ 100 µg/plate with S9 and at ≥ 3330 µg/plate without S9 and in TA1535 at doses of ≥ 100 µg/plate with S9 and at ≥ 1000 ug/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at all doses evaluated ≥ 333 µg/plate with and without S9.
FR-930 was re-evaluated in the confimatory mutagenicity assay under identical conditions, and similar results were observed (Trial 29938-C). Normal growth again was observed in all five tester strains evaluated with and without S9 except at 5000 µg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98 and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA100 at doses of ≥ 100 µg/plate with S9 and at ≥ 1000 µg/plate without S9 and in TA1535 at doses of ≥ 10.0 µg/plate with S9 and at ≥ 3330 µg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at the top 3 doses with and without S9. The mean positive control value for TA1537 without S9 did not meet the minimum protocol criteria for an acceptable positive control (3-fold increase over the vehicle control). For this reason, a repeat confirmatory assay was conducted in TA1537 with and without S9.
FR-930 was re-evaluated in a repeat confirmatory mutagenicity assay under identical conditions, and similar results to the initial mutagenicity assay were observed (Trial 29938-D1). Normal growth again was observed with and without S9 except at 5000 µg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in TA1537 with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures.
All positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

Conclusions:

SUMMARY:
The objective of this study is to evaluate the test article, FR-930, and/or its metabolites for their ability to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (Salmonella; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).
FR-930 was evaluated in the dose range-finding assay in tester strains TA100 and WP2uvrA. Ten doses of test article, from 6.67 to 5000 µg/plate, were evaluated with and without S9 (one plate per dose). No cytotoxicity was observed with either tester strain in the presence or absence of S9 as evidenced by no dose-related decreases in revertant frequency. Normal bacterial background lawns were observed up to 667 µg/plate with both tester strains in the absence of S9, and with TA100 in the presence of S9. Normal bacterial background lawns were observed up to 1000 µg/plate with WP2uvrA in the presence of S9. Test article precipitate was observed on the plates with both tester strains at doses ≥ 1000 µg/plate with and without S9. The test article precipitate obscured the bacterial background lawns at ≥ 1000 ug/plate ( ≥ 3330 pg/plate for WP2uvrA with S9). Dose-related increases in revertant frequency were observed with TA100 in the presence and absence of S9.
Based upon the results of the dose range-finding assay, FR-930 was evaluated in the initial mutagenicity assay, in all five tester strains, at doses of 1.00, 3.33, 10.0, 33.3, 100, 333, 1000 and 5000 µg/plate with S9 and 10.0, 33.3, 100, 333, 1000, 3330, and 5000 µg/plate without S9. All doses of the test article, as well as the concurrent positive and vehicle controls, were evaluated in triplicate plates. Normal growth again was observed in all five tester strains evaluated with and without S9 except at the highest 1-2 doses where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98, TA1537, and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA100 at doses of ≥ 100 µg/plate with S9 and at ≥ 3330 µg/plate without S9 and in TA1535 at doses of ≥ 100 µg/piate with S9 and at ≥ 1000 µg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at all doses evaluated ≥ 333 µg/plate with and without S9.
FR-930 was re-evaluated in the confirmatory mutagenicity assay under identical conditions, and similar results were observed. Normal growth again was observed in all five tester strains evaluated with and without S9 except at 5000 µg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98 and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA100 at doses of ≥ 100 µg/plate with S9 and at ≥3330 µg/plate without S9 and in TA1535 at doses of ≥ 100 µg/plate with S9 and at ≥ 1000 µg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at all doses evaluated ≥ 333 µg/plate with and without S9.

FR-930 was re-evaluated in the confirmatory mutagenicity assay under identical conditions, and similar results were observed. Normal growth again was observed in all five tester strains evaluated with and without S9 except at 5000 µg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in tester strains TA98 and WP2uvrA with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures. In contrast, dose-dependent increases in revertant frequencies were observed in TA100 at doses of ≥ 100 µg/plate with S9 and at ≥ 1000 µg/plate without S9 and in TA1535 at doses of ≥ 10.0 µg/plate with S9 and at ≥ 3330 µg/plate without S9. In addition, the test article again was found to be incompletely soluble in the aqueous top agar at the top 3 doses with and without S9. The mean positive control value fo TA1537 without S9 did not meet the minimum protocol criteria for an acceptable positive control (3-fold increase over the vehicle control). For this reason, a repeat confirmatory assay was conducted in TA1537 with and without S9.
FR-930 was re-evaluated in a repeat confirmatory mutagenicity assay under identical conditions, and similar results to the initial mutagenicity assay were observed. Normal growth again was observed with and without S9 except at 5000 µg/plate where the background lawns were obscured by precipitate. Revertant frequencies for all doses of FR-930 in TA1537 with and without S9 approximated or were less than those observed in the concurrent vehicle control cultures.
Except as noted, all positive and vehicle control values were within acceptable ranges, and all criteria for a valid study were met.

CONCLUSION:
These results indicate FR-930 was positive with tester strains TA100 and TA1535 both with and without S9 in the Bacterial Reverse Mutation Assay with a Confirmatory Assay under the conditions, and according to the criteria, of the test protocol.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test was performed according to a test protocol equivalent to OECD Guideline 474 (Mammalian erythrocyte micronucleus test). The study was conducted prior to the first approved version of the corresponding OECD guideline, thus is addressed in a weight of evidence approach in combination with the results of the in vitro studies. 

The objective of this test was to evaluate the test article for clastogenic activity in polychromatic erythrocyte (PCE) stem cells in treated mice. The route of administration was oral gavage, the most common route of administration for this test procedure. A subchronic dosing regimen was used: it consisted of two administrations approximately 24 hours apart. Treatment covered a 30-hour period or two cell cycles. Stock solutions of the test compound were prepared in dimethylsulfoxide (DMSO) at initial concentrations of 800 mg/ml and 100 mg/ml for administering the high (5 g/kg/day) and low (0.63 g/kg/day) doses respectively. The total doses were 10 g/Kg (highest dose) and 1.25 g/kg (lowest dose).

There was no evidence for induction of micronuclei by the test compound. In conclusion, the test item did not induce micronuclei at the doses tested.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1980

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

significant methodological deficiencies

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Significant deficiencies from current Guideline, e.g. samples were taken 6 hours after the last dose.

GLP compliance:

no

Type of assay:

mammalian erythrocyte micronucleus test

Specific details on test material used for the study:

Test material: Interstab FR 930

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Adult male and female mice, strain CD-1, from a randomly bred closed colony were purchased from Charles River Breeding Laboratories, Inc., Wilmington, MA. This healthy, random-bred strain has been selected to maximize genetic heterogeneity and at the same time assure access to a common source.

Animals were group-housed up to 15 mice per cage. A commercial diet (Purina Laboratory Chow) and water were offered ad libitum unless contraindicated by the particular experimental design.
Thirty-two outbred mice, 4 males and 4 females per dose level, were used. Animals were assigned to study groups at random according to Litton Bionetics, Inc. (LBI) Standard Operating Procedures (SOP). Prior to study initiation, animals were weighed to calculate dose levels according to SOP “Animal Weight Determinazion.” The volume of test article administrered per animal was established using this method unless there was significant variation among individuals, in which case individual calculations were made. Animals were uniquely identified by ear tag or ear punch. Dose or treatment groups were identified by cage card.

Sanitary cages and bedding was used. Personnel handling animals or working within the animal facilities were required to wear suitable protective garments. When appropriate, individuals with respiratory or other overt infections were excluded from the animal facilities.

Route of administration:

oral: gavage

Vehicle:

DMSO

Duration of treatment / exposure:

A subchronic dosing regimen was used. It consisted of two administrations approximately 24 hours apart. Treatment covered a 30-hour period or two cell cycles.

Frequency of treatment:

24h/dose

Dose / conc.:

10 000 mg/kg bw/day (actual dose received)

Remarks:

highest dose, FR 930

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

Remarks:

lowest dose, FR930

No. of animals per sex per dose:

4 males and 4 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Triethylenemelamine (TEM) at 1.0 mg/kg was dissolved in saline and used as the positive control article and was administered via a split dose intraperitoneal (IP) injection. The maximum amount administered to the experimental animals was 0.25 ml per male mouse and 0.20 ml per female mouse.

Tissues and cell types examined:

Mouse Bone Marrow

Details of tissue and slide preparation:

EXTRACTION OF BONE MARROW
Six hours after the last dose, animals were killed with CO2 and the adhering soft tissue and epiphyses of both tibia were removed. The marrow was aspirated from the bone and transferred to centrifuge tubes containing 5 ml fetal calf serum (one tube for each animal).

PREPARATION OF SLIDES
Following centrifugation to pellet the tissue, the supernatant was drawn off, cells resuspended in a drop of serum, and suspension spread on slides and air dried. The slides were then fixed in methanol, stained in May-Gruenwald solution followed by Giemsa, and rinsed in deionized water (Schmid, 1975).

SCREENING THE SLIDES
A thousand PCE’s per animal were scored. The frequency of micronucleated cells was expressed as percent micronucleated cells based on the total PCE’s present in the scored optic field. The normal frequency of micronuclei in this mouse strain in between 0.3 and 0.4%.

Evaluation criteria:

In tests performed for this evaluation only PCE’s were scored for micronuclei. Mature erythrocytes and other cells in the field were noted but not scored.

Two dose levels were established to ensure that a nontoxic level of the test article was scored. Dose response data are not necessary to define a test article as active. Responses considered positive are assumed to reflect clastogenic and related activities of test articles. Agents which break chromosomes and induce nondisjunction and other events which produce structural or numerical changes in chromosomes can produce micronuclei.

The data were analyzed by a student t-test (Brancroft 1957). Individual animal results were used as data points in the analysis. The set of micronuclei frequencies among the controls was compared to the set for each treatment level. Male and female animal data were analyzed separately and combined. Increases above the negative control frequency that are significant at p < 0.01 are considered indicative of an active agent.

For control of bias, all slides were coded prior to scoring and scored blind.

Key result

Sex:

male/female

Genotoxicity:

negative

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: did not induce micronuclei at the doses tested

Additional information on results:

The test material, Interstab FR930 is evaluated here for its ability to induce micronuclei in polychromatic erythrocytes (PCE, i.e. immature red blood cells) in mouse bone marrow.
The frequency of micronuclei in marrow from the negative control animals is within the normal range for this laboratory. The positive control compound, triethylenemelamine (TEM) induced a large increase in micronuclei, to a total of about 7%. There is no evidence for induction of micronuclei by the test compound.
Further details on results are present in the attache full study report.

Conclusions:

The test item Interstab FR930 did not induce micronuclei at the doses tested.

Executive summary:

The objective of this study was to evaluate the test article for clastogenic activity in polychromatic erythrocyte (PCE) stem cells in treated mice.

Adult male and female mice, strain CD-1, healthy and random-bred strain have been selected to maximize genetic heterogeneity and at the same time assure access to a common source.

Thirty-two outbred mice, 4 males and 4 females per dose level, were used. Prior to study initiation, animals were weighed to calculate dose levels according to SOP “Animal Weight Determinazion.” The volume of test article administrered per animal was established using this method unless there was significant variation among individuals, in which case individual calculations were made. 

The route of administration was oral gavage, the most common route of administration for this test procedure. A subchronic dosing regimen was used: it consisted of two administrations approximately 24 hours apart. Treatment covered a 30-hour period or two cell cycles. Stock solutions of the test compound were prepared in dimethylsulfoxide (DMSO) at initial concentrations of 800 mg/ml and 100 mg/ml for administering the high (5 g/kg/day) and low (0.63 g/kg/day) doses respectively. The total doses were 10 g/Kg (highest dose) and 1.25 g/kg (lowest dose).

The negative control article was administered by the same route as, and concurrently with, the test article and in volumes equal to the maximum amount administered to the experimental animals (0.25 ml per male mouse and 0.20 ml per female mouse).

Triethylenemelamine (TEM) at 1.0 mg/kg was dissolved in saline and used as the positive control article and was administered via a split dose intraperitoneal (IP) injection.

The frequency of micronuclei in mouse bone marrow from the negative control animals is within the normal range for this laboratory.

The positive control compound, triethylenemelamine (TEM) induced a large increase in micronuclei, to a total of about 7%.

There is no evidence for induction of micronuclei by the test compound. 

In conclusion, the test item did not induce micronuclei at the doses tested.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the available information, only the results of the in vitro bacterial gene mutation test indicated that the test item was positive with tester strains TA100 and TA1535 both with and without S9. 

The test item was studied further and did not show evidence of mutagenic activity to cultured human lymphocytes (both with and without metabolic activation) in any of the tests performed, was negative according to the mammalian cells gene mutation study and did not show transforming ability by the criteria of the CTAs performed and cannot be considered a potential carcinogen.

The reported in vitro information lend support to the conclusion that the test item is unlikely to represent a mutagenic hazard to man. 

Moreover, an in vivo micronucleus test was performed to evaluate the test item and there was no evidence for induction of micronuclei by the test compound. 

In conclusion, the test item did not show chromosomal and gene mutagenicity.