Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

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:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study. Meets generally accepted scientific standards, well documented and acceptable for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1981

Report date:

1981

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1983)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(adopted 1997)

Deviations:

yes

Remarks:

2-Aminoanthracene was used as a sole positive control in the presence of a metabolic activation system.

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

EC Number:

265-512-0

EC Name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Cas Number:

65140-91-2

Molecular formula:

C17 H29 O4 P. 1/2Ca

IUPAC Name:

calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Details on test material:

Purity: >98%
Physical state: solid

Method

Target gene:

Salmonella: HIS
E. coli: TRP

Species / strain

Species / strain / cell type:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA

Metabolic activation:

with and without

Metabolic activation system:

Aroclor1254 induced rat liver S9-Mix

Test concentrations with justification for top dose:

5, 10, 50, 100, 500, 1000 and 5000 µg/plate

Vehicle / solvent:

Methanol

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: With S9: 2-AA; Without S9: ENNG (TA100, TA1535, WP2UvrA), 2-NF (TA98, 1538), 9-AA (TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Each Petri dish contained:
- approx. 20 mL of minimum agar (Agar, Difco Laboratories, Detroit, Michigan, U.S.A., plus salts (Vogel-Bonner Medium E) and glucose)
- 0.1 mL of the solution of the test substance or the vehicle and 0.1 mL of a bacterial culture (in nutrient broth, Difco Laboratories, Detroit, Michigan, U.S.A., 0.8% plus 0.5% NaCl) in 2.0 mL of soft agar. The soft agar was composed of: 100 mL of 0.6% agar solution with 0.6% NaCl and 10 mL of a solution of L-histidine, 0.5 mM (Fluka, Buchs, Switzerland) and biotin 0.5 mM (Fluka, Buchs, Switzerland).
- In the experiments in which the substance was metabolically activated, 0.5 mL of an activation mixture was added also. One mL activation mixture contained: 0.1 mL S9 fraction of liver from rats induced with Aroclor 1254 (Analabs., Inc., North Haven, Connecticut, U.S.A.) and 0.9 mL of a solution of co-factor.

DURATION
- Exposure duration: 48h

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: background growth

POSITIVE CONTROLS:
With S9: 2-Aminoanthracene (2-AA); Without S9: N-ethyl-N´-nitro-N-nitroso guanidine (ENNG; TA100, TA1535, WP2UvrA), 2-nitrofluorene (2-NF; TA98, 1538), 9-Aminoacridine (9-AA; TA1537)

Evaluation criteria:

The test substance is generally considered to be non-mutagenic if the colony count in relation to the negative control is not doubled at any concentration.

Statistics:

When the colonies had been counted, the arithmetic mean was calculated.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the experiments performed with and without microsomal activation, comparison of the number of histidine- or tryptophanprototrophic mutants in the controls and after treatment with test substance revealed no marked differences (for details see "Any other information on results").

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments without microsomal activation.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538	E. coli WP2UVrA
0	33	170	16	8	12	22
5	29	151	13	7	10	24
10	29	159	14	8	12	28
50	30	147	16	11	7	30
100	29	145	18	9	14	26
500	33	152	22	7	11	28
1000	33	146	18	11	11	30
5000	34	146	16	8	15	33
ENNG
3		823	462			284
5		1603	67*			1225
2-NF
1	295				247
2	560				561
9-AA
40				47
80				791

*: A growth-inhibiting effect of the test substance was observed.

  Table 2: Number (arithmetic mean) of colonies of histidine-prototrophic back-mutants in experiments with microsomal activation.

Concentration (µg/plate)	TA98	TA100	TA1535	TA1537	TA1538	E. coli WP2UVrA
0	57	135	16	8	26	26
5	54	134	19	11	26	32
10	48	143	15	7	22	29
50	52	135	21	12	22	32
100	47	143	18	11	22	22
500	58	132	22	8	25	29
1000	43	140	18	4	16	36
5000	50	137	18	7	17	27
2-AA
0.5	132	163	122	41	110	1086
1.0	252	233	116	55	235	1063

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Based on the results of this study and under the conditions chosen, the test substance is considered as not mutagenic.

Executive summary:

In order to investigate the test article's potential to cause point mutation in bacteria, an AMES test similar in design to the OECD guideline No. 471 was carried out with the tester strains Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 and E. coli WP2 uvrA. The test article was applied by the plate incorporation method at concentrations of 5, 10, 50, 100, 500, 1000 and 5000 µg/0.1 ml either with or without a metabolic activation system (rat liver S9 mix). The experiment was performed in triplicates. Positive controls were performed in parallel to check the tester strains sensitivity. In none of the experiments did treatment with the test substance lead to an increase in the incidence of histidine-prototrophic mutants in comparison with the controls. In conclusion, no evidence of the induction of point mutations by the test substance or by its metabolites formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the test substance is considered as not mutagenic in this assay.