Ethanone, 2,2-dichloro-1-[5-(2-furanyl)-2,2-dimethyl-3-oxazolidinyl]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A number ofin vitro and in vivo studies were conducted, looking at potential mutagenicity of MON 13900 in various test systems. The results are summarised below:

In vitro

A study was conducted to determine the mutagenic potential of MON 13900 according to OECD Guideline 471 in compliance with GLP. A dose range-finding assay was conducted using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2uvrA in the presence and absence of an aroclor-induced rat liver metabolic activation system S9 (one plate per dose). Ten doses, from 6.67 to 5,000 µg/plate, were evaluated. Apparently, normal background growth was observed in both tester strains at all doses in the presence and absence of S9. In addition, the test substance was found to be freely soluble at all doses. Based upon these results, MON 13900 was evaluated in the mutagenicity assay in S. typhimurium strains TA98, TA100, TA1535 and TA1537, and E. coli strain WP2uvrAat 33.3, 100, 333, 1,000, 3,330 and 5,000 µg/plate with and without S9. Inhibited background growth was observed in TA98 and TA100 at 5,000 µg/plate without S9. Increases in revertant frequencies to approximately 1.6 - and 1.4 -fold the concurrent vehicle control values were observed in TA100 with and without S9, respectively. These increases appeared to be dose-dependent. Revertant frequencies in all other tester strains with and without S9 approximated or were lower than control values. The test substance was re-evaluated in an independent confirmatory assay under identical conditions, and similar results were observed. Except for strain TA1537 in the initial mutagenicity assay where the negative controls were above the acceptable ranges and a retest was performed, all positive and negative control data were within acceptable ranges and all criteria for a valid study were met. Thus, reproducible increases in revertant frequencies to approximately 1.4 - to 1.6 -fold control values were observed in strain TA100 with and without S9. In addition, these increases appeared to be dose-dependent and exceeded historical control values. However, these increases were only observed at 3,330 and/or 5,000 µg/plate, and sometimes only in the presence of reduced background lawns. Also, the increases did not reach the two-fold level required for a positive response and were within acceptable negative control ranges. In conclusion, MON 13900 produced an equivocal response in S. typhimurium strain TA100, both with and without microsomal activation, but was clearly negative in E. coli strain WP2uvrA and S. typhimurium strains TA98, TA1535 and TA1537.

A further study was conducted to determine the mutagenic potential of MON 13900 in an Ames mutagenicity assay according to EPA OPP 84 -2 in compliance with GLP. The test used S. typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 mix. In the screen, no toxicity was observed for TA100 in the presence or absence of S9 at levels up to 10 mg/plate. The test substance was observed to be insoluble at 10 mg/plate and some toxicity was observed in plate incorporation assays at this dose. Mutagenic activity was observed for the test substance towards test strain TA100 in the presence and absence of S9. The combination of statistical analyses of the initial and repeat test results indicated three treatments with revertants/plate values significantly greater than controls (p < 0.01) and significant positive dose-responses (p < 0.01). Based on these results, it appears that the activity is direct-acting and not dependent on the presence of an exogenous metabolic activation system. The observed response was very weak in terms of magnitude (2 -fold above controls) and compared to other agents that are positive in this assay. The potency of the response as calculated by the slope of the dose response curves gave a range of 0.003 - 0.004 revertants/nmole in the presence and absence of S9 mix for both initial and repeat assays. The repeat assay of TA98 in the absence of S9 mix indicated one treatment level with revertants/plate values significantly greater than controls (p < 0.01), with no positive dose-response. This result was not observed in the initial assay and was not reproducible in a retest run at a narrow dose range. Under the test conditions, MON 13900 elicited a very weak mutagenic response towards S. typhimurium strain TA 100 independent of the presence of a metabolic activation system. The response of greater than 2 - fold the control was only observed at a concentration which was toxic (10 mg/plate) and at which the test substance was insoluble. No reproducible mutagenic activity was observed for any of the other strains (TA 98, TA 1535 and TA 1537) in the presence or absence of metabolic activation.

The clastogenic potential of MON 13900 was evaluated in an in vitro cytogenic assay using human lymphocytes according to the OECD Guideline 473, EU Method B.10, EPA OPPTS 870.5375 as well as ICH Harmonised Tripartite Guideline S2A and S2B. The substance was tested over a range of concentrations, both in the presence and absence of S9 mix in two independent assays. In an initial experiment, cells were exposed to solvent and a range of concentrations from 50 to 500 µg/mL (the highest dose that did not induce cytotoxicity) for 3 h in the presence and absence of S9. In a second experiment, the range was from 50 to 500 µg/mL for 3 h in the presence of S9 and from 10 to 100 µg/mL for 20 h in the absence of S9. In both experiments, cells were harvested 20 h after the start of treatment. Dose-related reductions in mitotic activity and cytotoxic effects on chromosome morphology occurred in cultures from both experiments. Statistically significant reductions in mean mitotic activity, compared to the solvent controls, were observed in both experiments at the high concentrations, with and without S9. Statistically significant increases in the percentage of aberrant cells, above the solvent control values, were seen in both experiments in the presence and absence of S9. The positive control materials induced significant increases in the percentage of aberrant cells. Therefore, under the conditions of this assay system, MON 13900 was clastogenic to cultured human lymphocytes treated in vitro in the presence and absence of S9.

Finally, an in vitro study was performed to investigate the potential of MON 13900 to induce gene mutations at the HPRT locus in Chinese Hamster Ovary (CHO) cells according to the EU Method B.10, OECD Guideline 476 and EPA OPP 84 -2 in compliance with GLP. The test substance was initially tested at three concentrations in the absence or presence of varying concentrations of S9. A confirmatory experiment was run in the absence of S9 at 100, 200, 300, 500 and 800 µg/mL and in the presence of 5% S9 at 10, 20, 30, 40 and 60 µg/mL. All treatments were performed in triplicate. In the initial experiment, significant cytotoxicity was observed in the absence of S9 at 500 mg/mL. In the presence of 1, 2, 5 and 10% S9, significant cytotoxicity was observed at concentrations of 300, 100, 40 and 40 µg/mL, respectively. A statistically significant increase in the mutation frequency at 500 µg/mL was observed in the absence of S9. This response was considered to be due to an elevation in one of the duplicates and no statistically significant dose-response was observed. Results from the confirmatory experiment revealed that the test substance was significantly cytotoxic in the absence of S9 at a concentration of 800 µg/mL and in the presence of 5% S9 at 30 µg/mL. No statistically significant increases in mutant frequency were observed. In conclusion, the test substance was not considered to be mutagenic in CHO cells under the conditions of the assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 24, 1988 to Sep 26, 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

yes

Remarks:

No stability and analytical concentration of the test subsatnce was determined. However, these deviations should not significantly impact study results.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.1, 0.3, 1, 3 and 10 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation for TA 98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

With metabolic activation for TA 98

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without metabolic activation for TA 100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With metabolic activation for TA 100

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: Sodium nitrite

Remarks:

Without metabolic activation for TA 1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation for TA 1535

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Without metabolic activation for TA 1537

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation for TA 1537

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 48 h at 37±10⁰C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY
Revertant colonies for plates with more than 500 revertant colonies/plate were estimated by counting revertant colonies in several fields under a stereomicroscope and multiplying the counted colonies by a factor relating the total plate area to the area of the counted fields. Revertant colonies measured in this manner are calculated to not more than three significant figures. Revertant colonies on other plates, except as noted, were counted with an Artek Model 880 automatic colony counter or counted by visual examination (<10 revertants/plate).

Evaluation criteria:

Results were considered positive for a strain/microsome combination if revertants/plate values were significantly elevated over control values (p<0.01) at three treatments and there was a statistically significant dose response (p<0.01).

Statistics:

Statistical analysis was performed on plate incorporation assay results after transforming revertant/plate values as log10 (revertants/plate). Analysis included Bartlett’s test for homogeneity of variance and comparison of treatments with controls using within-levels pooled variance and a one-sided t-test. Grubbs’ test was performed to determine if outliers were present. Statistical significance of dose response was evaluated by regression analysis for log10 transformed doses and revertants/plate.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results of the statistical analyses of the plate incorporation assay results indicated that the test substance was mutagenic towards test strain TA100 in the presence and absence of S-9 mix. In combination, statistical analyses of both the initial and repeat assays for TA100 in the presence and absence of S-9 mix, indicated three treatments with revertants/plate values significantly greater than controls (p<0.01) and significant positive dose responses (p<0.01). The mutagenic response was very weak compared to other agents which are positive in this assay (2 fold over control values) and the high dose levels (3 and 10 mg/plate) were required to elicit a detectable response. The revertants/mg values calculated from the slope of the dose response curves were similar, with a range of 9.6-12.8 in the presence of S-9 and 10.9-16.1 in the absence of S-9 Mix. No mutagenic activity was indicated in these assays towards any of the other test strains used (TA98, TA1535, and TA1537). None of these other strain/microsome combinations indicated three treatments with revertants/plate values greater than controls (p<0.01) or significant dose responses (p<0.01). The repeat assay of TA98 in the absence of S-9 mix indicated one treatment with revertants/plate values significantly greater than controls (P<0.01) but with no positive dose response (P<0.01). This result was not observed in the initial assay and was not reproducible in a retest run at a narrow dose range.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

A toxicity screen was conducted using test strain TA100 with and without S-9 Mix. The test substance was not toxic at levels up to 10 mg/plate in the presence or absence of S-9 Mix. The test substance was observed to be insoluble at a level of 10 mg/plate. The maximum treatment level used in plate incorporation tests was 10 mg/plate. Some toxicity was observed in plate incorporation assays at the 10 mg/plate level. In the initial assay, toxicity was observed for TA98 and TA100 in the absence of S-9. In the repeat assays toxicity was seen for test strains TA100 in the presence and absence of S-9 mix and for TA1535 in the presence of S-9 mix.

Conclusions:

Under the conditions of this study, MON 13900 produced a very weak mutagenic response towards S. typhimurium strain TA 100 independent of the presence of a metabolic activation system. The response of greater than two fold the control was only observed at a concentration (10 mg/plate) which was toxic and at which the test substance was insoluble. The results in all other tester strain were considered to be uniformly negative.

Executive summary:

A study was conducted to determine the mutagenic potential of MON 13900 in an Ames mutagenicity assay according to EPA OPP 84-2 in compliance with GLP.

The test used S. typhimurium strains TA98, TA100, TA1535 and TA1537 in the presence and absence of S9 mix. In the screen, no toxicity was observed for TA100 in the presence or absence of S9 at levels up to 10 mg/plate. The test substance was observed to be insoluble at 10 mg/plate and some toxicity was observed in plate incorporation assays at this dose. Mutagenic activity was observed for the test substance towards test strain TA100 in the presence and absence of S9. The combination of statistical analyses of the initial and repeat test results indicated three treatments with revertants/plate values significantly greater than controls (p < 0.01) and significant positive dose-responses (p < 0.01). Based on these results, it appears that the activity is direct-acting and not dependent on the presence of an exogenous metabolic activation system. The observed response was very weak in terms of magnitude (2-fold above controls) and compared to other agents that are positive in this assay. The potency of the response as calculated by the slope of the dose response curves gave a range of 0.003 - 0.004 revertants/nmole in the presence and absence of S9 mix for both initial and repeat assays. The repeat assay of TA98 in the absence of S9 mix indicated one treatment level with revertants/plate values significantly greater than controls (p < 0.01), with no positive dose-response. This result was not observed in the initial assay and was not reproducible in a retest run at a narrow dose range.

Under the test conditions, MON 13900 elicited a very weak mutagenic response towards S. typhimurium strain TA 100 independent of the presence of a metabolic activation system. The response of greater than 2- fold the control was only observed at a concentration which was toxic (10 mg/plate) and at which the test substance was insoluble. No reproducible mutagenic activity was observed for any of the other strains (TA 98, TA 1535 and TA 1537) in the presence or absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 11 Sep, 2001 to Nov 5, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial gene mutation assay

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: The two additional mutations which enhance their sensitivity to some mutagenic compounds were uvrB gene and rfa wall mutation. Also, strains TA98 and TA100 contain the pKM101 plasmid.

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: One additional mutation which enhances their sensitivity to some mutagenic compounds was uvrA gene mutation.

Metabolic activation:

with and without

Metabolic activation system:

S9 homnogenate

Test concentrations with justification for top dose:

Dose-range finding study: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3330 and 5000 µg/plate
Mutagenecity assay: 33.3, 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble in DMSO.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo[a]pyrene (for TA98)

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene (for TA 98)

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for TA 100)

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (for TA 100)

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for TA 1535)

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (for TA 1535)

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for 1537)

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191 (for 1537)

Remarks:

Without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (for WP2uvrA)

Remarks:

With S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide (for WP2uvrA)

Remarks:

Without S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar (plate incorporation)

DURATION
- Exposure duration: 52 ± 4 h at 37 ± 2⁰C

NUMBER OF REPLICATIONS: Three

DETERMINATION OF CYTOTOXICITY: A minimum of three non-toxic doses were required to evaluate assay data. Cytotoxicity is detectable as a decrease in the number of revertant colonies per plate and/or by a thinning or disappearance of the bacterial background lawn. A slight thinning of the bacterial background lawn which is not accompanied by a reduction in the number of revertants per plate will not be evaluated as an indication of cytotoxicity. The condition of the bacterial background lawn was evaluated both macroscopically and microscopically (using a dissecting microscope) for indications of cytotoxicity and substance precipitate. Evidence of cytotoxicity was scored relative to the vehicle control plate and was recorded along with the revertant counts for all plates at that dose level. Lawns were scored as normal (N), reduced (R), obscured by precipitate (0), macroscopic precipitate present (P), absent (A), or enhanced (E); contaminated plates (C) also were noted.

Evaluation criteria:

Once the criteria for a valid assay had been met, responses observed in the assay were evaluated:
-Tester Strains TA98, TAI100 and WP2uvrA. For a test substance to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.
-Tester Strains TA1535 and TA 1537: For a test substance to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least one of these testers strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test substance.

Metabolic activation:

with and without

Genotoxicity:

other: Equivocal response in Salmonella strain TA100, both with and without microsomal activation, but was clearly negative in E. coli strain WP2uvrA and Salmonella strains TA98, TA1535 and TA1537.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Test substance handling: The test substance was stored at ambient temperature. The test substance was found to be insoluble in water at concentrations of 100 and 50.0 mg/mL and only slightly soluble in water at a concentration of 25.0 mg/mL with sonication (some of the test substance formed a heterogenous suspension). In contrast, the test substance formed a solution at a concentration of 101 mg/mL in dimethylsulfoxide (DMSO). For this reason, DMSO was selected as the vehicle. At 100 mg/mL, which was the most concentrated stock solution prepared for the mutagenicity assay; the test substance formed a transparent orange or dark orange solution. The test substance remained a solution at all succeeding dilutions prepared for the mutagenicity assay.
Dose range-finding assay: A dose rangefinding assay was conducted on the test substance using tester strains TA100 and WP2uvrA in the presence and absence of S9 (one plate per dose). Ten doses of test substance, from 6.67 to 5000 µg/plate, were evaluated. Apparently, normal growth was observed in both tester strains at all doses evaluated with and without S9. In addition, the test substance was found to be freely soluble at all doses evaluated.
Mutagenicity Assay: Based upon the results of the dose rangefinding study, the test substance was evaluated in the mutagenicity assay in all five tester strains at doses of 33.3, 100, 333, 1000, 3330 and 5000 µg/plate with and without S9. All doses of the test substance, as well as the concurrent positive and vehicle controls, were evaluated using three plates per dose. Inhibited background growth was observed in tester strains TA98 and TA100 at a dose of 5000 µg/plate without S9. In addition, the test substance was found to be freely soluble at all doses evaluated with and without S9. Increases in revertant frequencies, to approx 1.6- and 1.4-fold the concurrent vehicle control values, were observed in tester strain TA 100 with and without S9, respectively. In addition, these increases appeared to be dose dependent. Revertant frequencies in all other tester strains with and without S9 approximated or were less than control values. However, the mean values of the vehicle controls for tester strain TA 1537 with and without S9 were 101 and 82 revertants/plate, respectively. These values were above the acceptable ranges for this tester strain (2 to 25 revertants/plate). Therefore, the test substance was reevaluated in tester strain TA 1537 with and without S9. All other positive and negative control data were within acceptable ranges. The test substance was re-evaluated in an independent confirmatory assay under identical conditions, and similar results were observed. Inhibited background growth again was observed in tester strain TA100 at doses of ≥3330 µg/plate without S9, and the test substance again was freely soluble at all doses evaluated with and without S9. Increases in revertant frequencies, to approx 1.5- and 1.4-fold the concurrent vehicle control values, again were observed in tester strain TA100 with and without S9, respectively. In addition, these increases again appeared to be dose dependent. Revertant frequencies in all other tester strains with and without S9 approximated or were less than control values. All positive and negative control data were within acceptable ranges. In the further confirmation experiment, revertant frequencies in tester strain TA1537 with and without S9 approximated or were less than control value. All positive and negative control data were within acceptable ranges. All criteria for a valid study were met.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Mutagenicity results of the test substance inSalmonella strain TA100:

Dose/Plate (µg)	Mean Revertants Per Plate
	+S9		-S9
	Initial Assay	Confirmatory Assay	Initial Assay	Confirmatory Assay
Vehicle Control	110	117	95	101
33.3	89	118	98	75
100	101	120	104	100
333	114	116	95	99
1000	117	113	99	102
3330	153	140	135	114^a
5000	171	179^a	136	142^a
Positive Control	981	1179	1379	973

^aReduced background lawn

Conclusions:

Equivocal response in Salmonella strain TA100, both with and without microsomal activation, but was clearly negative in E. coli strain WP2uvrA and Salmonella strains TA98, TA1535 and TA1537.

Under the test conditions, MON 13900 produced an equivocal response in Salmonella strain TA100, both with and without microsomal activation, but was clearly negative in E. coli strain WP2uvrA and Salmonella strains TA98, TA1535 and TA1537.

Executive summary:

A study was conducted to determine the mutagenic potential of MON 13900 according to OECD Guideline 471 in compliance with GLP.

A dose range-finding assay was conducted using Salmonella typhimurium strain TA100 and Escherichia coli strain WP2uvrA in the presence and absence of an aroclor-induced rat liver metabolic activation system S9 (one plate per dose). Ten doses, from 6.67 to 5,000 µg/plate, were evaluated. Apparently normal background growth was observed in both tester strains at all doses in the presence and absence of S9. In addition, the test substance was found to be freely soluble at all doses. Based upon these results, MON 13900 was evaluated in the mutagenicity assay in S. typhimurium strains TA98, TA100, TA1535 and TA1537, and E. coli strain WP2uvrAat 33.3, 100, 333, 1,000, 3,330 and 5,000 µg/plate with and without S9. Inhibited background growth was observed in TA98 and TA100 at 5,000 µg/plate without S9. Increases in revertant frequencies to approximately 1.6- and 1.4-fold the concurrent vehicle control values were observed in TA100 with and without S9, respectively. These increases appeared to be dose-dependent. Revertant frequencies in all other tester strains with and without S9 approximated or were lower than control values. The test substance was re-evaluated in an independent confirmatory assay under identical conditions, and similar results were observed. Except for strain TA1537 in the initial mutagenicity assay where the negative controls were above the acceptable ranges and a retest was performed, all positive and negative control data were within acceptable ranges and all criteria for a valid study were met. Thus, reproducible increases in revertant frequencies to approximately 1.4- to 1.6-fold control values were observed in strain TA100 with and without S9. In addition, these increases appeared to be dose-dependent and exceeded historical control values. However, these increases were only observed at 3,330 and/or 5,000 µg/plate, and sometimes only in the presence of reduced background lawns. Also, the increases did not reach the two-fold level required for a positive response and were within acceptable negative control ranges.

In conclusion, MON 13900 produced an equivocal response in S. typhimurium strain TA100, both with and without microsomal activation, but was clearly negative in E. coli strain WP2uvrA and S. typhimurium strains TA98, TA1535 and TA1537. 

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 Dec, 1999 to 20 July, 2000

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2A

Qualifier:

according to guideline

Guideline:

other: ICH Harmonised Tripartite Guideline S2B

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Metabolic activation system:

Rat liver derived metabolic activation system

Test concentrations with justification for top dose:

Experiment 1: 50, 250 and 500 µg/mL
Experiment 2: 50, 250 and 500 µg/mL for 3-h treatment; 10, 50 and 100 µg/L for 20-h treatment

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: 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:

Experimental design: Duplicate human peripheral blood culture, were exposed to the solvent, test substance or positive control substances at appropriate concentrations in the following experiments
1. A cytogenetic experiment was conducted using a sample of pooled blood in the presence and absence of S9-mix. Cells were exposed to the test substance and control for a period of 3-h both in the presence and absence of S9-mix. Solvent, untreated and positive control cultures were included.
2. A second independent cytogenetic experiment was conducted using a sample pooled blood in the presence and absence of S9-mix. Cells were exposed to the test substance and control substances for a period of 3-h in the presence of S9-mix and 20-h in the absence of S9-mix. Solvent, untreated and positive control cultures were included. Treatment of the cultures started approx 48-h after culture initiation. A single sampling time, 20-h after the start of treatment was used. The sampling time of 20-h after the start of treatment used in this study was based on a measured mean cell cycle time for cultured human lymphocytes of 13.5-h. In both experiment, a range of concentration of the test substance was used in order to define suitable concentrations for chromosomal aberration analysis.

Culture establishment: Human blood samples were obtained by venepunture in lithium heparin tubes on the days of culture from healthy, non-smoking donors. Equal volume of blood from three females donors were polled together for each experiment. All donors have a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes. At 0-h, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 tissue culture medium supplemented with approx 10% foetal bovine serum (FBS), 1.0 IU/mL heparin, 100 IU/mL penicillin, and 100 µg/mL streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (5% v/v) and were maintained at 37⁰C for 48-h with gentle mixing.

Metabolic activation system: The metabolic activation system was prepared as a 1:1 mixture of S9 fraction and cofactor solution. S9 was prepared from male SD rats dosed once daily by oral gavage for 3 d with a combined phenobarbital and β-napthoflavone corn-oil preparation. The treated animals were sacrificed on the day following the third dose. A 25% w/v homogenate was prepared. The co-factor solution was prepared as a single stock solution of Na2HPO4, KCl, glucose-6-phosphate NADP (Na salt) and MgCl2 in sterile double deionized water and adjusted to a final pH of 7.4.

Culture treatment: Prior to treatment, the cultures to be treated for a 20-h period were centrifuged and the culture medium was replaced with fresh supplemented RPMI-1640 culture medium. The cultures to be treated for a 3-h period did not receive a medium change at this point. Approx 48-h after culture establishment, aliquots of the test substance, solvent control or positive controls were administered to duplicate cultures. In addition, 200 µL of a 1:1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. Cultures from both experiments in the presence of S9-mix and cultures from experiment 1 in absence of S9-mix were treated for a period of 3-h at 37⁰C, after which the culture medium was removed following centrifugation and replaced with fresh supplemented medium. The cultures were re-incubated at 37⁰C for a remainder of the 68-h growth period. Cultures from experiment 2 in the absence of S9-mix were treated for a period of 20-h until, the end of the 68-h growth period. The effect of the test substance on the pH and osmolality of the culture medium was investigated using single cultures containing medium only as changes in pH and increases in osmolality have been reported to result in the production on chromosomal aberrations.

Culture harvesting: Approx 2-h prior to harvesting the cultures were treated with colcimid. 68-h after the culture establishment the culture were centrifuges, the supernatant was removed and the cells were re-suspended in approx 10 mL of 0.075 KCl at room temperature for approx 10 min. The cultures were centrifuged, the supernatant was removed and the remaining cells fixed in freshly prepared metnanol/glacial acetic acid fixtative added dropwise and make up to a volume of approx 10 mL. The fixative was removed following centrifugation and replaced with freshly prepared fixative. After at least 2 subsequent changes of foxtative, slides were made by dropping the cell suspension on to clean moist labeled microscope slides. The slides were air dried, stained in filtered Giemsa stain double ionized water for min, rinsed in water, air-dried and mounted with cover slip in DPX.

Evaluation criteria:

The percentage of aberrant metaphases was calculated for each treatment scored both including and excluding cells with only gap-type aberrations. The Fischer Exact Probability test (one-sided) was used to evaluate statistically the percentage or metaphase showing aberration (excluding cell with only gap-type aberrations). Data from each treatment group in the presence and absence or S9-mix was compared with the respective solvent control group values. The data was interpreted as follows:
1. No statistically significant increase in the percentage or aberrant cells at any concentration above concurrent solvent control values –Negative.
2. A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values which falls within the laboratory solvent control range-Negative.
3. An increase in the percentage of aberrant cells at least at one concentration which is substantially greater than the laboratory historical solvent control value-Positive.
4. A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described above may require further evaluation.

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Determination of mitotic indices and selection of concentrations: The highest concentration selected for chromosomal aberration was 500 µg/mL following a 3-h treatment period and 100 µg/mL following a 20-h treatment period. Concentration related reduction in mitotic activity and cytotoxic effects on the chromosomal morphology were observed in cultures from both experiments, thus demonstrating that the test substance is biologically active in this test system. Significant reductions in mean mitotic activity, compared to the solvent control values, were observed in cultures from both experiments treated with the highest concentration of the test substance selected for chromosomal aberration analysis. Cultures treated in higher concentration of the test substance were considered not to be suitable for chromosomal aberration analysis due to lack of metaphase as a result of toxicity or severe cytotoxic effects on chromosome structure. No significant effect on osmolality was observed.

Results of chromosomal aberration analyses: Statistically significant increase in the percentage of aberrant cells, above the solvent control values were recorded in cultures in experiment 1 and 2 treated with the test substance in the presence and absence of S9-mix.

Table 1. In vitro cytogenetics assay: Results in the absence of S9:

Treatment	Concentration µg/mL	# of Cells Scored	% Cells with Structural Aberrations^a	Average Mitotic Index
Experiment 1
Solvent control	0	200	3.0	20.15
Test substance	50	200	2.5	19.30
	250	200	15.5^**	15.65
	500	100	42.0^**	11.15
Positive control	0.5	25	60.0^**	15.70
Experiment 2
Solvent control	0	200	1.5	12.50
MON 13900	10	200	3.0	11.30
	50	200	8.5^**	11.00
	100	100	26.0^**	8.25
Positive control (MC)	0.2	25	36.0^**	7.90

^aExcluding gaps

^**p ≤0.01 (Fischer’s exact test)

Table 2. In vitro cytogenetics assay: Results in the presence of S9

Treatment	Concentration µg/ml	# of Cells Scored	% Cells with Structural Aberrations^a	Average Mitotic Index
Experiment 1
Solvent control	0	200	2.0	20.00
MON 13900	50	200	1.5	17.30
	250	200	10.5^**	11.55
	500	100	41.0^**	9.20
Positive control	50	25	48.0^**	14.40
Experiment 2
Solvent control	0	200	0.5	8.20
Test substance	50	200	2.5	9.90
	250	200	6.5^**	9.00
	500	100	26.0^**	8.25
Positive control (CP)	50	25	36.0^**	8.60

^aExcluding gaps

^**p ≤0.01 (Fischer’s exact test)

Conclusions:

Under the test conditions, MON 13900 was clastogenic to cultured human lymphocytes in vitro in the presence and absence of S-9.

Executive summary:

The clastogenic potential of MON 13900 was determined in anin vitrocytogenic assay using human lymphocytes according to the OECD Guideline 473, EU Method B.10, EPA OPPTS 870.5375 as well as ICH Harmonised Tripartite Guideline S2A and S2B.

The substance was tested over a range of concentrations, both in the presence and absence of S9 mix in two independent assays. In an initial experiment, cells were exposed to solvent and a range of concentrations from 50 to 500 µg/mL (the highest dose that did not induce cytotoxicity) for 3 h in the presence and absence of S9. In a second experiment, the range was from 50 to 500 µg/mL for 3 h in the presence of S9 and from 10 to 100 µg/mL for 20 h in the absence of S9. In both experiments, cells were harvested 20 h after the start of treatment. Dose-related reductions in mitotic activity and cytotoxic effects on chromosome morphology occurred in cultures from both experiments. Statistically significant reductions in mean mitotic activity, compared to the solvent controls, were observed in both experiments at the high concentrations, with and without S9. Statistically significant increases in the percentage of aberrant cells, above the solvent control values, were seen in both experiments in the presence and absence of S9. The positive control materials induced significant increases in the percentage of aberrant cells.

Therefore, under the conditions of this assay system, MON 13900 was clastogenic to cultured human lymphocytes treatedin vitroin the presence and absence of S9.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Remarks:

(with the few exceptions: concentrations and stability in carrier were not determined by analytical method. Solutions were prepared on each day of use. These deviations were had no impact on the quality or integrity of the study)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

Selective agent employed: 6-thioguanine

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Initial experiment: 0, 100, 300, 500 µg/mL (without S9); 0, 50, 100, 300 µg/mL (with 1% S9); 0, 20, 50, 100 µg/mL (with 2% S9); 0, 10, 30, 40 µg/mL (with 5% S9); 0, 20, 30, 40 µg/mL (with 10% S9)
Confirmatory experiment: 0, 100, 200, 300, 500, 800 µg/mL (without S9); 0, 10, 20, 30, 40 and 60 µg/mL (with 5% S9)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ethylmethane sulphonate (200 µg/mL)

Remarks:

in the absence of S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Benzo(a)pyrene (1 µg/mL)

Remarks:

in the presence of S9 mix

Details on test system and experimental conditions:

All treatments were performed in triplicate. Cultures were exposed to the test substance for 3 h at 37⁰C, both in the presence and absence of metabolic activation. The expression period was 7-9 d.

Species / strain:

Chinese hamster Ovary (CHO)

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

Remarks on result:

other: strain/cell type: Chinese hamster Ovary (CHO)

Remarks:

Migrated from field 'Test system'.

Table 1: CHO HGPRT gene mutation assay: Initial mutagenicity determination of MON 13900 at different concentrations of S9:

Treatment	Concentration (µg/mL)	Cytotoxicity (relative survival)	Mutagenicity (mean mutant frequency x 10^6)
No S9	0	-	4.6
	100	0.83	21.2
	300	0.63	15.2
	500	0.54	45.9*
1% S9	0	-	6.1
	50	0.87	8.3
	100	0.66	8.9
	300	0.33	22.6
2% S9	0	-	16.5
	20	0.79	9.8
	50	0.65	9.5
	100	0.03	0.0
5% S9	0	-	17.1
	10	0.94	12.9
	30	0.62	28.1
	40	0.40	9.1
10% S9	0	-	12.5
	20	1.05	2.4
	30	0.63	7.1
	40	0.09	3.4
*p<0.05

 

Table 2. CHO HGPRT gene mutation assay: Confirmatory mutagenicity determination of MON 13900 in the absence and presence of S9 (5%):

Treatment	Concentration (µg/ml)	Cytotoxicity (relative survival)	Mutagenicity (mean mutant frequency x 10^6)
No S9	0	-	5.6
	100	1.01	5.4
	200	0.96	11.4
	300	0.83	6.2
	500	0.61	17.2
	800	0.38	2.6
5% S9	0	-	2.7
	10	0.82	3.6
	20	0.55	4.8
	30	0.25	4.1
	40	0.16	17.6
	50	0.06	5.0

Conclusions:

MON 13900 was not mutagenic in CHO cells under the conditions employed in this assay.

Executive summary:

An in vitro study was performed to investigate the potential of MON 13900 to induce gene mutations at the HPRT locus in Chinese Hamster Ovary (CHO) cells according to the EU Method B.10, OECD Guideline 476 and EPA OPP 84-2 in compliance with GLP.

The test substance was initially tested at three concentrations in the absence or presence of varying concentrations of S9. A confirmatory experiment was run in the absence of S9 at 100, 200, 300, 500 and 800 µg/mL and in the presence of 5% S9 at 10, 20, 30, 40 and 60 µg/mL. All treatments were performed in triplicate. In the initial experiment, significant cytotoxicity was observed in the absence of S9 at 500 mg/mL. In the presence of 1, 2, 5 and 10% S9, significant cytotoxicity was observed at concentrations of 300, 100, 40 and 40 µg/mL, respectively. A statistically significant increase in the mutation frequency at 500 µg/mL was observed in the absence of S9. This response was considered to be due to an elevation in one of the duplicates and no statistically significant dose-response was observed. Results from the confirmatory experiment revealed that the test substance was significantly cytotoxic in the absence of S9 at a concentration of 800 µg/mL and in the presence of 5% S9 at 30 µg/mL. No statistically significant increases in mutant frequency were observed.

In conclusion, the test substance was not considered to be mutagenic in CHO cells under the conditions of the assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

A number ofin vitroandin vivostudies were conducted, looking at potential mutagenicity of MON 13900 in various test systems. The results are summarised below:

In vivo

MON 13900 was assessed for potential clastogenicity to rat bone marrow cellsin vivoaccording to the OECD Guideline 475 in compliance with GLP. In a dose range-finding study, the test article was mixed with the vehicle control, corn oil, at dose levels of 200, 500, 600, 800, 1,500 and 2 000 mg/kg bw and 500, 800, 1,200, 1,500 and 2,000 mg/kg bw for males and females, respectively, and administered once via the oral route to three animals per designated dose level. Based on the findings of the dose range-finding study, dose levels of 0, 150, 300 and 600 mg/kg bw were selected for testing in the chromosome aberration assay in males only. Colchicine was administered 1.5 to 2.5 h before sacrifice for the two harvest time points; 18 and 42 h. The positive control was cyclophosphamide dissolved in sterile deionized water (60 mg/kg bw harvest at only 18 h). If available, 100 cells/animal were examined from five of the six rats/dose level. At least 25 metaphase cells were analyzed from animals that had 25% of the cells with one or more aberrations. Numbers and types of chromosomal aberrations, mitotic index and vernier location of each metaphase containing damage were recorded. The mitotic index was recorded as the number of cells in mitosis per 1,000 cells counted. Mortality and clinical observations were observed in the 600 mg/kg bw group at the 18 h harvest time point. The clinical signs observed in the high dose rats included hypoactivity, hunched posture, fecal staining and/or squinting eyes. One incidence each of fecal staining and rough haircoat was observed in the 300 mg/kg bw (mid dose) rats. There were no statistically significant increases in structural chromosomal aberrations or numerical chromosome changes. Under the conditions of this study, the test substance was not considered to be clastogenic to rat bone marrow cellsin vivoat any of the dose levels tested.

A study was conducted to evaluate the potential of MON 13900 to induce genetic damage by measuring DNA repair as unscheduled DNA synthesis (UDS) in primary hepatocyte cultures from treated male Sprague-Dawley rats according to OECD Guideline 486 in compliance with GLP. Male rats (4 to 6 per group) were administered single doses of the test substance at 0, 50, 150 and 300 mg/kg bw, vehicle or positive control by gavage. Hepatocytes were isolated 2 and 16 h after dosing, radiolabeled with³H-methyl-thymidine and evaluated for UDS. In addition, an evaluation of serum enzymes and liver histopathology was conducted at 16 h to assess potential hepatic toxicity. All animals survived the duration of the study and o clinical signs of toxicity were noted. Increased serum levels of the liver enzymes AST and/or ALT were observed in one or more animals from each of the test substance dose groups. Mean AST and ALT activities at the high dose were increased 2.2- and 3.6-fold, respectively, relative to vehicle controls. Histopathological findings considered related to administration of the test substance were hepatic leukocytic infiltration, increased hepatic eosinophilia and enlarged portal hepatocytes in rats that received 150 or 300 mg/kg bw test substance. No effects were noted at 50 mg/kg bw. No evidence of UDS was noted in any of the treatment groups. Positive responses were seen with the positive controls dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2‑AAF). The percent of cells in repair (%IR) for the 2-AAF positive control group was slightly below the criteria specified by the protocol (16vs.20%) but within the laboratory’s historical range for positive controls. Furthermore, the 20% IR criteria was based on historical control data for DMN, not for 2-AAF, for which far fewer laboratory historical control data existed. This deviation was not considered to have impacted the acceptability of the results of this study. Under the test conditions, hepatotoxicity but no evidence of unscheduled DNA synthesis (UDS) was observed in male rats following administration of a single oral dose of the test substance.

Finally, a study was designed to determine the potential of MON 13900 to induce chromosome effects in anin vivomouse bone marrow micronucleus assay according to a protocol equivalent or similar to EU Method B12, US EPA 84-2 and OECD Guideline 474 in compliance with GLP. The test substance was administered intraperitoneally (i.p.) to three groups of CD-1 mice (15 males and 15 females/dose) at 0, 47.5, 95 and 190 mg/kg bw (maximum dose equivalent to approximately 98% of the LD50) (males) and 37.5, 75 and 150 mg/kg bw (maximum dose equivalent to approximately 92% of the LD50) (females). Groups of five males and five females were sacrificed at 24, 48 and 72 h post-dosing. A concurrent positive control was dosed with cyclophosphamide (i.p., 60 mg/kg bw) and sacrificed at 24 h post-dosing. Bone marrow smears were prepared from all animals, fixed, stained and examined microscopically. At least 1,000 erythrocytes per animal were scored for micronuclei. 2/15 males and 2/15 females died at dose levels of 190 and 150 mg/kg bw, respectively. Hypoactivity was observed in both sexes at these dose levels (up to the 48 and 72 h time point in males and females, respectively). Statistically significant (p < 0.05) decreases in mean body weight were observed in animals from the high dose group at 48 and 24 h. No decreases in meanpolychromatic erythrocytes (PCE)/total erythrocyte ratios were observed, indicating the absence of bone marrow toxicity. A statistically significant (p < 0.05) increase in the meanmicronucleated polychromatic erythrocytes(MNPCE) frequency was observed at 24 h in males receiving 95 mg/kg bw. No statistically significant increases were noted in any of the other treatment groups. This isolated increase was not considered to be treatment-related because there was no effect in males at 190 mg/kg bw or in females from all treated groups. In conclusion, the test substance did not induce chromosome effectsin vivoin mouse bone marrow cells under the experimental conditions of this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 4 Oct, 1999 to 19 Oct, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

other: Crl:CD”(SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult male and female rats of the Crl:CD”(SD)IGS BR strain were purchased from Charles River Laboratories, Raleigh, NC. This is an outbred strain that maximizes genetic heterogeneity and therefore tends to eliminate strain-specific response to test substances. The animals were acclimated for at least 7 d before being placed on study. The animals were housed in sanitary, stainless-steel, hanging, wire cages. The animals were housed, separated by gender, up to two animals per cage during acclimation, and individually after randomization.
The animals were housed under the following climatic conditions: temperature, 64 to 79°F; humidity, 30 to 70%; light cycle, 12 h light/dark; air changes, at least 10 air per h. A commercial diet, PMI@Feeds, Inc. Certified Rodent Diet # 5002 (chow), and tap water were available ad libitum. The feed was analyzed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates, and specified nutrients. The water was analyzed biannually, on a retrospective basis, for specified microorganisms, pesticides, heavy metals, alkalinity, and halogens. The animals were randomly assigned, by a computer program, to study dose groups. Each animal was uniquely identified by ear tag. Treatment groups were identified by cage label. The animals were weighed prior to dosing and dosed based upon the individual animal weights. The weight variation of the animals did not exceed ±20% of the mean weight of each sex. All animals were dosed on an acute (one-time only) basis. Unscheduled deaths, if any, were discarded without necropsy.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Justification for choice of solvent/vehicle: The test substance is soluble in corn oil
-- Concentration of test material in vehicle: 7.5, 15 and 30 mg/mL
- Dosing volume: 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prior to dosing, the test substance was ground with a mortar and pestle for approx 5 min. Each level of the test substance was prepared by adding the appropriate volume of the vehicle, corn oil, to a premeasured quantity of the test article. The formulations were mixed until in suspension. The stock concentrations of 7.5, 15 and 30 mg/mL were prepared for dose level of dose levels of 150, 300, and 600 mg/kg bw, respectively.

Duration of treatment / exposure:

One time only

Frequency of treatment:

One time only

Remarks:

Doses / Concentrations:
0, 150, 300 and 600 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

Dose range-finding study: 3 males and/or females
Main assay: 6 males per dose level at the 24 h harvesting time, and 6 males at the high-dose and control levels at the 48 h harvesting time. 10 replacement males were included at the high-dose level as potential replacements for original high-dose animals.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide

Tissues and cell types examined:

Bone marow cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Based on the findings of the dose rangefinding study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Five animals at each dose level, and five vehicle and positive control animals were euthanized approximately 18 hours after dosing for extraction of the bone marrow. At approximately 42 hour after dosing, five animals dosed at the 600 mg/kg dose level and five animals dosed with the vehicle control article were euthanized for extraction of the bone marrow.

DETAILS OF SLIDE PREPARATION: Approx 1.5 to 2.5 h prior to euthanasia, the animals were injected intraperitoneally with 2.0 mg/kg of colchicines (10 mL/kg). The hind limb bones (tibias and femurs) were removed from the first five surviving animals, when possible, for marrow extraction. The marrow was flushed from the bone and transferred to Hanks’ balanced salt solution. The marrow pellet was collected by centrifugation and then resuspended in 0.075M KCl. The centrifugation was repeated and the pellet was resuspended in fixative (methanol:acetic acid, 3:1). The fixative was then changed and the fixed cells were dropped onto glass slides and air dried. The metaphase cells were stained with 5% Giemsa and air dried; the slides were coverslipped. Animals in excess of the first five survivors (according to eat-tag sequence) were euthanized and no marrow was extracted.

METHOD OF ANALYSIS: The first five surviving animals in each treatment and control group were analyzed, when possible. Slides were coded for control of bias and scored for chromosomal aberrations as defined in the following section. Cells were selected for scoring on the basis of good chromosome morphology, and only cells with the number of centromeres equal to the rat modal number 42±2 were analyzed. Normally 100 cells from each animal, if available, were read. At least 25 metaphase cells were analyzed from animals that have 25% of the cells with one or more aberrations. For each cell bearing an aberration, the microscope stage location (vernier readings) was recorded so that the cell may be relocated if necessary. Percent polyploidy and endoreduplication were also analyzed by evaluating 100 metaphases per animal, if available, and tabulated. A mitotic index was calculated by scoring the number of cells in mitosis per 1000 cells observed, and generating a percentage.

Evaluation criteria:

Gaps were not counted as significant aberrations. Open breaks were considered as indicators of genetic damage, as were configurations resulting from the repair of breaks. The latter included translocations, multiradials, rings, multicentrics, etc. Cells with more than one aberration was considered to indicate more genetic damage than those containing evidence of single events. Frequently, one is unable to locate 100 suitable metaphase spreads for each animal even after preparing additional slides. Possible causes for this may be related to cytotoxic effects which may alter the duration of the cell cycle, kill the cell, or cause clumping of the chromosomes. Animals with less than 25 analyzable cells were not included in the statistical analysis. Additional information was gained from the mitotic index which also appeared to reflect cytotoxic effects. Comparison with a concurrent vehicle control which happens to show an unusually low frequency of aberrations may suggest statistical significance which is not biologically relevant.
In these instances, the treatment data were considered against the historical control database.
Analyses were performed on a per animal basis for the following variables:
1) Number of cells with a least one structural aberration
2) Number of cells with two or more structural aberrations

Statistics:

Analysis of variance (ANOVA) techniques were used to compare positive control to the vehicle control group. Specifically, Levene’s test was performed to test for variance homogeneity. In the case of variance heterogeneity, the data were ranked. Dunnett’s test was performed to compare test article-treated group means to the vehicle control. In this case, additional tests such as linear regression and Terpstra-Jonckheere test for monotone trend were also performed to evaluate any possible dose-response. The type of aberration, its frequency, the statistical significance of any increase and its correlation to dose in a given time period were all considered in evaluating the clastogenic potential of the test article. The criterion for a positive response is generally a statistically significant dose-related increase in the number of structural aberrations for at least one dose level.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Clinical signs in the high-dose animals included, but were not limited to, hypoactivity, hunched posture, fecal staining, and/or squinted eyes.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose range-finding assay:
-Dose selection: Dose levels of 200, 500, 600, 800, 1500, and 2000 mg/kg bw and 500, 800, 1200, 1500, and 2000 mg/kg bw were chosen for males and females, respectively, in the dose range-finding assay.
-Dosing information: Thirty-five animals, approx 8 wk old at the time of dosing, with a weight range of 227 - 252 g and 163 - 200 g, for the males and females, respectively, were assigned to this study. Two animals were replaced due to misdosing caused by backwashing. At the termination of this assay, all surviving animals were euthanized by CO, inhalation followed by incision of the diaphragm. The treatment regimen for this assay is shown in Table 1. Prior to dosing, the test substance was groundwith a mortar and pestle for approx 5 to 10 min. Each level of the test substance was prepared by adding the appropriate volumeof the vehicle, corn oil, to a premeasured quantity of the test substance. The formulations were mixed until in suspension. The stock concentrations and descriptions are shown in the Table 2.
-Results and interpretation: The mortality data for this assay are summarized in Table 3. Based on these results, the maximum tolerated dose was estimated to be 600 mg/kg bw.

Chromosome aberration assay:
-Dose selection: Based on results from the dose range-finding assay, dose levels of 150,300, and 600 mg/kg bw were selected for testing in rats in this assay. Only males were used in the chromosome aberration assay because there were no substantial differences in clinical observations between the sexes in the dose range-finding assay. A replacement dose group consisting of 10 animals was included at the top dose level in anticipation of mortality.
-Dosing information: 52 animals, approx 8 wk old at the time of dosing, with a weight range of 245 - 300 g were used in this assay. An outline of the dosing scheme and harvest timepoints is found in table 4. Cyclophosphamide, the positive control, was dissolved in sterile deionized water. Prior to dosing, the test substance was ground with a mortar and pestle for approx 5 min. Each level of the test substance was prepared by adding the appropriate volume of the vehicle, corn oil, to a premeasured quantity of the test substance. The formulations were mixed until in suspension. The stock concentrations and descriptions are shown in table 5.
-Animal observations: All animals were examined immediately after dosing, about 1 h after dosing, and at least daily for the duration of this assay for toxic signs and/or mortalities. All animals in the vehicle and positive control groups appeared normal after dosing and remained healthy until the appropriate harvest timepoints. Few signs of toxicity such as fecal stains, convulsions, hypoactive were also observed.
-Results and interpretation: The test substance induced mortality and signs of clinical toxicity in the high-dose (600 mg/kg) animals by the 18 h harvest time point. Clinical signs in the high-dose animals included, but were not limited to, hypoactivity, hunched posture, fecal staining, and/or squinted eyes. One incidence each of fecal staining and rough haircoat was noted in the mid-dose (300 mg/kg) animals. There were no statistically significant increases in either structural chromosome aberrations or numerical chromosome changes. The test substance was therefore concluded to be negative for induction of structural or numerical chromosome damage under the conditions of exposure in this assay.

Analytical results: A 24 h room temperature stability analysis was performed on the 2 mg/mL sample prepared by Analytical Chemistry and yielded a % change of 3.60%. A 24 h room temperature stability analysis was performed on the 30.00 mg/mL Trial 1 sample prepared by Genetic Toxicology and yielded a % change of -1.60%. There were no interfering peaks detected at the retention time of the peak of interest in either control analyzed for stability.

Conclusions:

Under the conditions of this study, MON 13900 was not considered to be clastogenic to rat bone marrow cells in vivo at any of the dose levels tested.

Executive summary:

A study was conducted to assess the clastogenic potential of MON 13900 to rat bone marrow cellsin vivoaccording to the OECD Guideline 475 in compliance with GLP.

In a dose range-finding study, the test article was mixed with the vehicle control, corn oil, at dose levels of 200, 500, 600, 800, 1,500 and 2 000 mg/kg bw and 500, 800, 1,200, 1,500 and 2,000 mg/kg bw for males and females, respectively, and administered once via the oral route to three animals per designated dose level. Based on the findings of the dose range-finding study, dose levels of 0, 150, 300 and 600 mg/kg bw were selected for testing in the chromosome aberration assay in males only. Colchicine was administered 1.5 to 2.5 h before sacrifice for the two harvest time points; 18 and 42 h. The positive control was cyclophosphamide dissolved in sterile deionized water (60 mg/kg bw harvest at only 18 h). If available, 100 cells/animal were examined from five of the six rats/dose level. At least 25 metaphase cells were analyzed from animals that had 25% of the cells with one or more aberrations. Numbers and types of chromosomal aberrations, mitotic index and vernier location of each metaphase containing damage were recorded. The mitotic index was recorded as the number of cells in mitosis per 1000 cells counted. Mortality and clinical observations were observed in the 600 mg/kg bw group at the 18 h harvest time point. The clinical signs observed in the high dose rats included hypoactivity, hunched posture, fecal staining and/or squinting eyes. One incidence each of fecal staining and rough haircoat was observed in the 300 mg/kg bw (mid dose) rats. There were no statistically significant increases in structural chromosomal aberrations or numerical chromosome changes. 

Under the conditions of this study, the test substance was not considered to be clastogenic to rat bone marrow cellsin vivoat any of the dose levels tested.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Characterization, homogeneity, stability of the of test and control substances was not conducted/determined. These deviations were not considered to have had an impact on the quality and integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

Characterization, homogeneity, stability of the of test and control substances was not conducted/determined. These deviations were not considered to have had an impact on the quality and integrity of the study.

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 84-2

Deviations:

yes

Remarks:

Characterization, homogeneity, stability of the of test and control substances was not conducted/determined. These deviations were not considered to have had an impact on the quality and integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals used in this study were seven to twelve week old male and female CD-1 mice (Source: Charles River Laboratories Inc., Portage, MI). Upon receipt, the animals were quarantined for a minimum of 7 d. Only animals considered to be normal were released from quarantine and used for testing. Prior to testing, the mice were uniquely identified using ear tags and corresponding cage cards. The animals were housed two per cage prior to dosing and one per cage after dosing. The animals were housed in stainless steel cages with stainless steel mesh bottoms. Animals were selected for the different test (or control) groups by a computer generated randomization scheme. Water was provided ad libitum via an automatic watering system. Purina Certified Laboratory Rodent Chow@ No. 5002 was used as the diet and was provided ad Iibitum. This diet has been determined to be nutritionally acceptable for the maintenance of laboratory rodents and has been certified by the manufacturer not to contain contaminants likely to interfere with the study. The animals were housed in rooms designed to routinely maintain a 12-h light cycle, temperature range of 64-79°F, and relative humidity of 40-70%. There were no excursions in animal room environmental conditions which had any obvious impact on the results of the study. Animal housing and husbandry were in accordance with the provisions of the ‘Guide for the Care and Use of Laboratory Animals’, USPHS-NIH Publication No. 86-23.

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil

Details on exposure:

Solutions or suspensions of the test substance were prepared on the day of use, using corn oil as the vehicle. Animals were treated by a single intraperitoneal injection of corn oil (vehicle control, 10 mL/kg body weight), test substance in corn oil (10 mL of solution/kg bw) or cyclophosphamide in Hank’s balanced salt solution (positive control, 10 mL of solution/kg body weight). The positive control used was commercial grade cyclophosphamide monohydrate.

Duration of treatment / exposure:

Single injection

Remarks:

Doses / Concentrations:
0, 47.5, 95 and 190 mg/kg bw for males and 0, 37.5, 75 and 150 mg/kg bw for females
Basis:
nominal conc.

No. of animals per sex per dose:

15 males and 15 females/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Intraperitoneal injection
- Doses / concentrations: 10 mL of solution/kg bw

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

Extraction of bone marrow cells sand slide preparation: All animals were sacrificed by cervical dislocation and their femora were removed. Each bone was opened at the end and the bone marrow was flushed with approx 2 mL of fetal bovine serum into a centrifuge tube. Bone marrows from the femora of each animal were pooled for slide preparation. The suspension was centrifuged to remove the serum. Portions of the remaining cells were placed on a clean glass microscope slide and a smear was prepared. Two slides were prepared for each sample and the remaining cell suspension was stored refrigerated to prepare additional slides, if necessary. Following preparation of the smears the slides were allowed to air dry overnight. The slides were stained using a Hems-Tek II slide staining machine and a Wright-Giemsa Stain Pak which includes stain, buffer and rinse solutions.

Scoring of Slides: Slides of bone marrow cells were coded prior to distribution and slides were scored without knowledge of the treatment or control group to which the slides belonged. For each animal, two scorers evaluated: a) 500 total erythrocytes for polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) and, b) 500 PCEs for micronucleated polychromatic erythrocytes (MN PCEs). PCEs and NCEs were distinguished by different staining properties’. Micronuclei were identified as uniform, darkly stained, round or oval shaped bodies found in the cytoplasm of PCEs. Bodies in PCEs which were refractive, improperly shaped or stained, or which were not in the focal plane of the cell, were not scored as micronuclei. PCES containing more than one micronucleus were scored as a single micronucleated PCE. Scoring data were used to calculate, for each animal, the ratio of PCES per total erythrocytes (PCEs plus NCEs) and the number of MN PCEs per 1000 PCEs.

Evaluation criteria:

To determine whether a statistically significant response in micronucleated polychromatic erythrocytes (MN PCE) frequency is treatment related the following criteria were considered: (a) whether there are dose and time-dependent effects that are consistent with a treatment-induced response and (b) the degree of the response in relation to both concurrent and historical negative and positive control data.

Statistics:

LD50 estimates were calculated using the probit method on toxicity range-finding data. The individual test animal was used as the individual unit for analysis of micronucleated PCE frequency, PCE/total erythrocyte ratio and body weight change. Micronucleated PCE frequencies observed for each animal were transformed as the square root prior to analysis. PCE/totaI erythrocyte ratios were not transformed. A Dunnett’s test (one sided) was used for comparison of treatment group and positive control values with vehicle control values. A critical value of p≤0.05 was used for statistical significance.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Hypoactivity and decrease in the body weight

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 100 to 1000 mg/kg bw
- Clinical signs of toxicity in test animals: Hypoactivity and mortality


RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): No
- Induction of micronuclei (for Micronucleus assay): No
- Ratio of PCE/NCE (for Micronucleus assay): No

The test substance was toxic to the male mice dosed at the high dose level (190 mg/kg bw) as evidenced by clinical signs of hypoactivity up to the 48-h time point. Two male mice of the high dose level were found dead, one at 24-h and one at 72 -h after dosing. The test substance was toxic to the female mice dosed at the high dose level (150 mg/kg bw) as evidenced by clinical signs of hypoactivity up to the 72-h time point. Two female mice of the high dose level were found dead 48 -h after dosing. Statistically significant decreases in mean body weight were observed for the high dose level male group sacrificed 48-h after dosing and for the high dose level female group sacrificed 24-h after dosing. No deaths, clinical signs or significant decreases in mean body weight were observed in any of the other test substance treated groups or control groups (vehicle and positive control).

Conclusions:

MON 13900 did not induce chromosome effects in vivo in mouse bone marrow cells under the experimental conditions of this study.

Executive summary:

A study was conducted to evaluate the potential of MON 13900 to induce chromosome effects in anin vivomouse bone marrow micronucleus assay according to the guideline equivalent or similar to EU Method B12, US EPA 84-2 and OECD Guideline 474 in compliance with GLP.

The test substance was administered intraperitoneally (i.p.) to three groups of CD-1 mice (15 males and 15 females/dose) at 0, 47.5, 95 and 190 mg/kg bw (maximum dose equivalent to approximately 98% of the LD50) (males) and 37.5, 75 and 150 mg/kg bw (maximum dose equivalent to approximately 92% of the LD50) (females). Groups of five males and five females were sacrificed at 24, 48 and 72 h post-dosing. A concurrent positive control was dosed with cyclophosphamide (i.p., 60 mg/kg bw) and sacrificed at 24 h post-dosing. Bone marrow smears were prepared from all animals, fixed, stained and examined microscopically. At least 1,000 erythrocytes per animal were scored for micronuclei. 2/15 males and 2/15 females died at dose levels of 190 and 150 mg/kg bw, respectively.  Hypoactivity was observed in both sexes at these dose levels (up to the 48 and 72 h time point in males and females, respectively). Statistically significant (p < 0.05) decreases in mean body weight were observed in animals from the high dose group at 48 and 24 h. No decreases in mean polychromatic erythrocytes (PCE)/total erythrocyte ratios were observed, indicating the absence of bone marrow toxicity. A statistically significant (p < 0.05) increase in the meanmicronucleated polychromatic erythrocytes(MNPCE) frequency was observed at 24 h in males receiving 95 mg/kg bw. No statistically significant increases were noted in any of the other treatment groups. This isolated increase was not considered to be treatment-related because there was no effect in males at 190 mg/kg bw or in females from all treated groups.

In conclusion, the test substance did not induce chromosome effects in vivo in mouse bone marrow cells under the experimental conditions of this study.

Reference 3

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From Aug 9, 2001 to Feb 5, 2002

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)

Deviations:

yes

Remarks:

Dosing solutions were not analyzed.

GLP compliance:

yes (incl. QA statement)

Type of assay:

unscheduled DNA synthesis

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

Supplier: Charles River Laboratories, Kingston, Area K97
Weights at Study Initiation: 262-335 g
Number of Animals: 46
Age at Study Initiation of Experiment: 8-9 wks
Weight Range at Dose Administration: All animals were weighed on the day of dosing. Mean animal weights in the experiment fell within the Protocol specified range of 150 – 400 g. All animals assigned to test weighed within ±20% of the mean. Individual body weights are not reported, but are maintained in the data and used only to determine dosage.
Animal Care: General procedures for animal care and housing were in accordance with the NRC Guide for the Care and Use of Laboratory Animals (1996) and the Animal Welfare Standards incorporated in 9 CFR Part 3, 199 1.
Quarantine: 5 d
Housing: 4-5/cage
Cages: Clear polycarbonate
Bedding: Hardwood chip
Light Cycle: Approx 12 h light/ 12 h dark; a deviation occurred when animals were exposed to ~5 additional h of light in order to dose the 16 h time point.
Temperature: 70⁰ to 72⁰F
Humidity: 54-56%
Food: Purina Certified Rodent Chow, #5002, ad libitum, (PMI Nutrition International). No contaminants which could interfere and affect the results of the study were present in the food, based on an analysis report from the supplier.
Water: Purified (filtered, deionized, and UV-treated) tap water, ad libitum. No contaminants which could interfere and affect the results of the study were present in the water based on analysis reports.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Corn oil
- Concentration of test material in vehicle: 10 mL/kg
- Type and concentration of dispersant aid (if powder):
- Lot/batch no. (if required): 070K0127

Details on exposure:

-Preparation of dose formulations: The test substances for the high dose was mixed with the vehicle the day prior to dosing and allowed to stir overnight. The next morning particles were observed suspended in solution. At this time the lower dose formulations were made by serial dilution from the high dose after allowing to mix ~10 min between dilutions. Each dose was left mixing at room temperature prior to dosing. Preparation of the high dose formulation the day prior to dosing deviated from the study protocol which indicates formulating on the day of dosing. However, the high dose was prepared earlier to allow mixing overnight, and dosed the morning of the next day all within a 24 h period.
-Characterization of dose formulations: All unused dosing formulations were stored at -20⁰C until the end of the study. No analysis was conducted.

Duration of treatment / exposure:

Single oral dose administration at either an early (2 h) or late (12-16 h) time point

Remarks:

Doses / Concentrations:
0, 50, 150 or 300 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

4 to 6 rats per group

Control animals:

yes, concurrent vehicle

Positive control(s):

Dimethylnitrosamine (DMIN), 2-acetylaminofluorene (2-AAF)
- Justification for choice of positive control(s): NMR spectrum consistent with structure
- Route of administration: Gavage
- Doses / concentrations: 10 mL/kg

Tissues and cell types examined:

Hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The high dose for the UDS assay of 300 mg/kg was established by the Sponsor based on the results of previous studies with the test substance. The mid and low doses for the UDS assay were designated as 150 mg/kg and 50 mg/kg, respectively, to demonstrate a potential dose response.
Necropsy:
-Interval: Approx 2.5 and 16 h post-dose
Anesthesia/Euthanasia: Sodium pentobarbital was administered intraperitoneally to cause anesthesia (~ 65 mg/kg). Animals did not recover from surgery. Extra animals not assigned to the study were euthanized with an overdose of sodium pentobarbital.
-Blood Collection and Analysis: Approx 16 h post-dose -2 mL of blood was collected by catheterizing the hepatic portal vein prior to liver perfusion. Collected samples were allowed to sit for ~30 min at room temperature, then centrifuged at 3000 rpm for 15 min to isolate serum. Each serum sample was pipetted into a secondary container and shipped on wet ice to IDEXX
Veterinary Services (IDEXX) for analysis of alanine aminotransferase (ALT), alkaline phosphatase (ALP), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH).
-Tissues Retained: Hepatocyte biopsies were collected on the 16 h control and test substsnce treatment groups. Approx 16 h post-dose and after the blood sample was collected, the distal end of the frontal liver lobe was clamped off with a hemostat. The end of the lobe was then removed and placed into neutral buffered formalin.
-Histopathology: Sections of tissue were embedded in paraffin and cut approximately 5 µm thick, attached to slides, and stained with hematoxylin and eosin. All processed tissues were examined microscopically by a board certified pathologist.
Hepatocyte Isolation:
Animals were anesthetized with sodium pentobarbital prior to surgery and did not recover from surgery. Primary cell cultures were obtained from the livers of treated and control animals as described below. Livers were perfused using a collagenase solution. Isolated hepatocytes were combed out of the perfused liver and inoculated into 6-well culture dishes (containing coverslips) in Williams' Medium E (WE) supplemented with 2 mM 1-glutamine, 50 [1g/mL gentamicin sulfate, and 10% fetal bovine serum. After ~1. 5 h of incubation in a humidified atmosphere at 37⁰C, -5% C0 2, the cultures were washed to remove nonviable cells (those not attached to the coverslips). All washes and subsequent culturing were done in serum-free media. Cultures were incubated in WE containing 10 [µCi/mL 3H-thymidine (specific activity, ~80 Ci/mmole) for approx 4 h at 37⁰C, ~ 5% CO2, followed by ~14 to 20 h in WE containing 0.25 mM unlabeled thymidine.
Cell culture fixation and staining: Cultures were washed, swelled in 1% sodium citrate, fixed in 1:3 glacial acetic acid:ethanol, and washed with deionized water. The coverslips with attached cells were mounted to slides, allowed to dry, and 3 slides from each treatment were visually selected based on attachment for autoradiography. Selected slides were dipped in Kodak NTB-2 emulsion, and exposed at -20⁰C for 7 d prior to development. Cells were stained with 1% methyl-green Pyronin Y.
Evaluation of slides: Slides were evaluated under a light microscope for quality of attachment, pyknotic cells, or obvious morphologic changes and stained following the autoradiographic procedures. The best slides were then selected for UDS scoring and coded by a person not scoring the experiment. Random letter codes were generated by a random letter generator program on an IBM-compatible PC (VI VOBOOK version 3.01).

Evaluation criteria:

The test substance is considered positive if the mean net grain (NG) count for any group is greater than 0 and if the percent of cells in repair (%IR) is greater than 20%.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Biochemical changes and hepatotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Increased serum levels of aspartate (AST) and/or alanine (ALT) aminotransferases were observed in one or more animals from each of the tested groups. Mean AST and ALT activities in the 300 mg/kg dose group were increased 2.2- and 3.6-fold, respectively, relative to the vehicle controls. These increases were not statistically significant due to the large animal to animal variability and low number of animals evaluated. However, these increases were considered to be toxicologically significant. No variation from control values were seen for either alkaline phosphatase or lactate dehydrogenase, the only other clinical chemistry parameters evaluated. Histopathological findings include hepatic leukocyte infiltration, increased hepatocytic eosinophilia, and enlarged portal hepatocytes in rats that received 150 mg/kg and 300 mg/kg test substance. These lesions were found in 3 of 5 (60%) of the animals at 150 mg/kg, and in 3 of 6 (50%) to 5 of 6 (83%) of the animals at 300 mg/kg. No similar changes were present in the liver sections of rats that received either the vehicle control or 50 mg/kg test substance.

Unscheduled DNA synthesis (UDS) in male rats:

Treatment	Dose (mg/kg)	2-Hours		16-Hours
		NG	%IR	NG	%IR
MON 13900	0	-5.5	1	-3.8	3
	50	-3.4	3	-5.2	0
	150	-5.8	0	-6.9	0
	300	-6.0	0	-6.2	0
DMN	10	8.9	56	-	-
2-AAF	10	-	-	0.8	16

Conclusions:

Under the test conditions, hepatotoxicity but no evidence of unscheduled DNA synthesis (UDS) was observed in male rats following administration of a single oral dose of MON 13900.

Executive summary:

A study was conducted to evaluate the potential of MON 13900 to induce genetic damage by measuring DNA repair as unscheduled DNA synthesis (UDS) in primary hepatocyte cultures from treated male Sprague-Dawley rats according to OECD Guideline 486 in compliance with GLP.

Male rats (4 to 6 per group) were administered single doses of the test substance at 0, 50, 150 and 300 mg/kg bw, vehicle or positive control by gavage. Hepatocytes were isolated 2 and 16 h after dosing, radiolabeled with³H-methyl-thymidine and evaluated for UDS. In addition, an evaluation of serum enzymes and liver histopathology was conducted at 16 h to assess potential hepatic toxicity. All animals survived the duration of the study and o clinical signs of toxicity were noted. Increased serum levels of the liver enzymes AST and/or ALT were observed in one or more animals from each of the test substance dose groups. Mean AST and ALT activities at the high dose were increased 2.2- and 3.6-fold, respectively, relative to vehicle controls. Histopathological findings considered related to administration of the test substance were hepatic leukocytic infiltration, increased hepatic eosinophilia and enlarged portal hepatocytes in rats that received 150 or 300 mg/kg bw test substance. No effects were noted at 50 mg/kg bw. No evidence of UDS was noted in any of the treatment groups. Positive responses were seen with the positive controls dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2‑AAF). The percent of cells in repair (%IR) for the 2-AAF positive control group was slightly below the criteria specified by the protocol (16vs.20%) but within the laboratory’s historical range for positive controls. Furthermore, the 20% IR criteria was based on historical control data for DMN, not for 2-AAF, for which far fewer laboratory historical control data existed. This deviation was not considered to have impacted the acceptability of the results of this study.

Under the test conditions, hepatotoxicity but no evidence of unscheduled DNA synthesis (UDS) was observed in male rats following administration of a single oral dose of the test substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on results from in vivo testing, MON 13900 is not considered to be genotoxic and therefore does not require classification for this endpoint according to CLP (EC 1272/2008) criteria.