3,3'-methylenebis[5-methyloxazolidine]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 10, 2000 to September 17, 2000 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study, well performed acoording to good scientific standards

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

CONTRAM ™MBO

IUPAC Name:

CONTRAM ™MBO

Details on test material:

- Name of test material (as cited in study report): OS 157339
- Common name: CONTRAM ™MBO
- Substance type: Formaldehyde releaser
- Composition of test material, percentage of components: Reaction product from paraformaldehyde and 2 hydroxypropylamine (ratio of 3:2)
- Physical state: Pale yellow liquid
- Analytical purity: No data, Sponsor's responsibility
- Lot/batch No.: OS 157339
- Expiration date of the lot/batch: No data, Sponsor's respondibility
- Stability under test conditions: No data
- Storage condition of test material: At room temperature in the dark

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Type and identity of media: Top agar (Difco) overlaid onto Vogel-Bonner minimal agar plate
- Properly maintained: yes
- Periodically checked for sterility: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

other: Histidine deficiency S. typhimurium and tryptophan deficiency in E. coli

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 mix; male rats induced with oral phenobarbital and beta-naphthoflavone (80/100 mg/kg bw) on 3 consecutive days prior to S9-preparation.

Test concentrations with justification for top dose:

Vehicle (destilled water) control and 5, 15, 50, 150, 500, 1500 µg/plate in experiment 1; in experiment 2 TA100 (with MA) and TA98 (with and without MA) exposed to 5, 15, 50, 150, 300, 500 µg/plate and WP2uvrA (with and without MA) to 5, 15, 50, 150, 300, 500, 750 µg/plate.

Vehicle / solvent:

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

- Justification for choice of solvent/vehicle: : Test item had good solubility with water

Controls

Untreated negative controls:

other: In separate trials untreated control data given

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: N-ethyl-N’-nitro-N-nitrosoguanidine, 9-aminoacridine , 4-nitroquinoline-1-oxide, 2-aminoanthracene and benzo(a)pyrene

Remarks:

Without metabolic activation (MA) N-ethyl-N’-nitro-N-nitrosoguanidine: TA100, TA1535, WP2uvrA; 9-aminoacridine: TA1537; 4-nitroquinoline-1-oxide: TA98. With MA:2-aminoanthracene: TA100, TA1535 and TA1537, WP2uvrA; benzo(a)pyrene: TA 98

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Preincubation period: 10 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 replicates per dose, 2 independent experiments; vehicle (sterile distilled water) control and in separate trials untreated control data given.

NUMBER OF CELLS EVALUATED: Revertant colonies scored manually.

DETERMINATION OF CYTOTOXICITY
relative total growth

Evaluation criteria:

The test item is considered positive if in at least one strain a dose-related and reproducible statistically significant increase in the number of revertant counts in obtained.

Statistics:

Dunnett's method of linear regression

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Preliminary range finding study with & without MA at 0.15-5000 µg/plate (10 dose levels) in TA 100 and E. coli WP2uvrA.

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In preliminary study toxic effects at 500 µg/plate in TA100 and at 1500 µg/plate in E.coli (with and without MA)

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Without metabolic activation

Valid negative and positive controls (also comparison with historical control data of this lab). No significant increases in revertants were observed in TA1535 and TA1537 at any dose level without MA (not shown in the Table). A dose-related, reproducible and statistically significant increase in the frequency of revertants was detected in TA98, TA100 (only 2^ndexperiment), and WP2uvrA. However, the increase was only modest (see discussion below).

With metabolic activation

Valid negative and positive controls (also comparison with historical control data of this lab). No significant increases in revertants were observed in TA1535 and TA1537 at any dose level with MA (not shown in the Table). A dose-related, reproducible and statistically significant increase in the frequency of revertants were detected in TA98, TA100, and WP2uvrA (see Table above). However, the increase was only modest (see discussion below).

Table for number of revertant colonies in Experiment I
Average of 3 plates & standard deviation; historical control data 1998 and 1999

Concentration in µg/plate	WP2uvrA^- + MA	WP2uvrA^- -MA	TA98 +MA	TA98 -MA	TA100 +MA	TA100 -MA
vehicle control	14+-3.8	15+-4.4	29+-2.3	16+-4.6	140+-10	128+-11
historical con-trol (range)	25 (13-41) 27 (11-46)	23 (13-34) 23 (10-52)	36 (22-54) 31 (12-56)	31 (15-45) 24 (10-46)	125 (81-170) 107 (63-186)	124 (73-173) 102 (57-179)
5	20+-6.7	18+-2.1	27+-5.0	17+-5.0	132+-9	124+-11
15	20+-3.5	20+-4.6	28+-3.8	17+-4.6	142+-13	135+-9
50	28+-3.5**	18+-7.5	32+-5.5	21+-8.5	142+-1	132+-15
150	29+-2.1**	19+-6.4	46+-4.4**	32+-3.1**	258+-16**	135+-5
500	31+-7.2**	34+-6.7**	3+-1.5 V	0 V	0 V	0 V
1500	3+-1.5 V	0 V	0 T	0 T	0 T	0 T
positive control	300+-19	614+-59	310+-13	163+-5	1519+-194	479+-39

Number of revertant colonies (average of 3 plates) in Experiment II Average of 3 plates & standard deviation
Concentration in µg/plate	WP2uvrA- + MA	WP2uvrA- -MA	TA98 +MA	TA98 -MA	TA100 +MA	TA100 -MA
vehicle control	19+-6.1	18+-4.2	41+-1.5	27+-5.6	132+-17	95+-18
historical control (range)	25 (13-41) 27 (11-46)	23 (13-34) 23 (10-52)	36 (22-54) 31 (12-56)	31 (15-45) 24 (10-46)	125 (81-170) 107 (63-186)	124 (73-173) 102 (57-179)
5						72+-3
15	22+-4.6	19+-4.4	39+-1.5	26+-10.0	115+-2	74+-7
50	21+-0.6	22+-6.8	47+-8.1	26+-4.5	139+-21	75+-3
100			53+-12.7	36+-17	159+-6*
150	26+-8.1	23+-7.0	57+-4.6*	31+-10.6	219+-14**	142+-6.5**
300	41+-2.9**	35+-2.9**	19+-2.6 V	12+-1.7 S	82+-7 S
500	44+-8.1	29+-2.9*	0 V	0 V	0 V	0 V
750	23+-3.6 S	0 V
1500						0 T
positive control	760+-89	430+-21	212+-21	153+-12	1438+-235	452+-35
MA: metabolic activation; *: p<0.05; **:p<0.005; S: sparse bacterial background lawn; V: very weak bacterial background lawn; T: no bacterial background lawn; nd: not done

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
positive

Under the experimental conditions described in this study the test substance induce gene mutation in bacteria even at non-cytotoxic concentrations. Authors concluded that the test substance was considered to be mutagenic. But the increase in revertants reached max. a 2-fold of the current control. Comparing the results with the historical control data of the same laboratory the detected increased in revertants were within the control range except TA100+MA in Exp.I and TA98 in Exp.II (but no 2-fold increase in comparison to the vehicle control, see Table above). Statistically significance in comparison with the concurrent control did not necessarily means also toxicological relevance. However, the effects were dose-dependent and reproducible.

Executive summary:

Study according to OECD guideline 471(adopted 1997). Salmonella typhimurium reverse mutation test in TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2uvrA at concentration levels of 0, 5, 15, 50, 150, 300, 500, 750, 1500 µg/plate with and without metabolic activation. The test item showed a weak positive, reproducible response.