6H-Dibenz[c,e][1,2]oxaphosphorin-6-propanoic acid, butyl ester, 6-oxide

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:

January - April 2014

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

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:

butyl 3-{9-oxo-8-oxa-9λ⁵-phosphatricyclo[8.4.0.0²,⁷]tetradeca-1(14),2(7),3,5,10,12-hexaen-9-yl}propanoate

Cas Number:

848820-98-4

Molecular formula:

C19H21O4P

IUPAC Name:

butyl 3-{9-oxo-8-oxa-9λ⁵-phosphatricyclo[8.4.0.0²,⁷]tetradeca-1(14),2(7),3,5,10,12-hexaen-9-yl}propanoate

Constituent 2

Reference substance name:

805-659-5

EC Number:

805-659-5

IUPAC Name:

805-659-5

Test material form:

liquid: viscous

Details on test material:

Name: KCCS DOB11
Batch no.: DOB11-1301
Test material form: yellow to brown viscous liquid
Composition: 6H-Dibenz[c,e][1,2]oxaphosphorin-6-propanoic acid, butyl ester, 6-oxide (DOB11) ca. 97.7%; 10-Hydroxy-9,10-Dihydro-9-oxa-10-phosphaphenantrene-10-oxide (DOPO-Ox) ca. 0.7%; Butylacrylat ca. 0.2%; o-Cyclohexylphenol ca. 0.1%; others unknown
CAS No.: 848820-98-4
EINECS-No.: 805-659-5
Molecular formula: C19H21O4P
Molecular weight: 344.34 g/mol
Purity: 97.7% GC
Homogeneity: homogenous liquid
Production date: 12. Dec. 2013
Expiry date: 12. Dec. 2014
Storage: Room Temperature: (20 ± 5°C)

Method

Species / strain

Species / strain / cell type:

other: TA 97a, TA 100, TA 102 and TA 1535

Metabolic activation:

with and without

Metabolic activation system:

S9 (obtained by Trinova Biochem. Gießen, Batch no 3198; Specification produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally)

Vehicle / solvent:

A stock solution containing 50 g/L of the test item in dimethyl sulfoxide (DMSO) was prepared. DMSO was chosen as vehicle, because the test item was sufficiently soluble, and this solvent doesn’t have any effects on the viability of the bacteria or the number of spontaneous revertants.

Controls

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine, CAS-No.: 99-56-9

Details on test system and experimental conditions:

Test System
Specification
Species Salmonella typhimurium LT2
Strains TA 1535, TA 97a, TA 98, TA 100 and TA 102
Mutations TA97a: hisD6610, uvrB, pKM 101, rfa
TA 98: hisD3052, uvrB, pKM 101, rfa
TA 100: hisG46, uvrB, pKM 101, rfa
TA102: hisG428, pKM 101, rfa
TA1535: hisG46, uvrB, rfa

Salmonella typhimurium (all strains used) were obtained from TRINOVA BioChem (date of arrival: 20. Feb. 2013) and were stored as lyophilisate in the fridge at 2-8 °C. The lyophilisates were used to prepare permanent cultures which were filled into vials and stored at < -75 °C.
One day before the start of each experiment, one vial per strain to be used was taken from the deep freezer. The surface was scraped with an inoculation loop and the aliquot was put into a culture vessel containing nutrient broth. After incubation over night at 37 °C, the cultures were used in the experiment. During the test, the cultures were stored at room temperature as to prevent changes in the titre.

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

First Experiment

Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic mutagenes) showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity

The test item was dissolved in DMSO.

No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Therefore the test item is stated as not mutagenic under the test conditions.

To verify this result, a second experiment was performed using the pre-incubation method.

Survey of the Findings

The mean revertant values of the four replicates are presented in the following table (Mean Revertants First Experiment)

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	109	126	17	15	108	112	252	230	18	21
	sd	6.5	10.6	1.6	2.5	14.3	7.4	25.2	22.8	3.3	2.2
DMSO	Mean	133	132	13	17	155	125	213	241	20	21
	sd	29.2	27.4	3.0	1.7	33.9	8.3	15.9	20.4	3.9	4.4
Positive Controls*	Mean	704	520	222	154	778	680	997	962	280	341
	sd	39	15	35	25	54	42	38	111	40	29
	f(I)	5.29	3.94	17.08	9.06	7.20	5.44	4.68	3.99	15.56	16.24
5000 µg/pl.	Mean	107	102	12	10	104	142	244	218	23	19
	sd	8	12	2	0	5	11	10	34	2	3
	f(I)	0.80	0.77	0.92	0.59	0.67	1.14	1.15	0.90	1.15	0.90
1500 µg/pl.	Mean	137	101	10	16	137	140	253	240	22	21
	sd	7	10	0	3	5	8	5	34	4	5
	f(I)	1.03	0.77	0.77	0.94	0.88	1.12	1.19	1.00	1.10	1.00
500 µg/pl.	Mean	115	113	12	14	137	101	226	228	19	19
	sd	8	9	2	2	2	9	17	18	4	3
	f(I)	0.86	0.86	0.92	0.82	0.88	0.81	1.06	0.95	0.95	0.90
150 µg/pl.	Mean	118	134	14	13	118	135	226	225	19	20
	sd	17	15	3	3	17	30	44	25	3	1
	f(I)	0.89	1.02	1.08	0.76	0.76	1.08	1.06	0.93	0.95	0.95
50 µg/pl.	Mean	140	106	12	15	155	117	207	225	19	21
	sd	35	4	3	3	7	37	25	18	3	3
	f(I)	1.05	0.80	0.92	0.88	1.00	0.94	0.97	0.93	0.95	1.00

f(I) = increase factor

* Different positive controls were used

Second Experiment

Confirmation of the Criteria and Validity

The treatments for the sterility control and the determination of the titre didn’t show any inconsistencies. The determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory. All positive controls showed mutagenic effects with and without metabolic activation.

Solubility and Toxicity

The test item was dissolved in DMSO.

No signs of toxicity towards the tested strains could be observed. The background lawn was visible and the number of revertant colonies was not significantly reduced.

Mutagenicity

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation was observed. No concentration-related increase over the tested range was found.

Therefore the test item is stated as not mutagenic under the test conditions.

Survey of the Findings

The mean revertant values of the four replicates are presented in the following table (Mean Revertants Second Experiment)       

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
H2O	Mean	123	117	13	14	120	122	241	218	18	20
	sd	33.3	2.5	2.5	1.7	31.6	13.3	18.6	16.5	1.7	0.6
DMSO	Mean	127	150	15	14	108	128	252	241	21	21
	sd	24.5	20.0	2.4	3.5	10.8	16.4	15.0	33.0	4.4	3.6
Positive Controls*	Mean	361	375	219	175	732	1068	1128	999	226	103
	sd	40	38	12	15	157	69	145	65	31	13
	f(I)	2.84	2.50	14.60	12.50	6.10	8.34	4.48	4.15	12.56	4.90
5000 µg/pl.	Mean	128	107	15	11	109	117	243	222	19	18
	sd	11	11	3	2	7	36	29	27	5	3
	f(I)	1.01	0.71	1.00	0.79	1.01	0.91	0.96	0.92	0.90	0.86
2500 µg/pl.	Mean	137	118	14	14	124	160	222	236	18	16
	sd	5	4	2	1	14	6	22	38	2	0
	f(I)	1.08	0.79	0.93	1.00	1.15	1.25	0.88	0.98	0.86	0.76
1250 µg/pl.	Mean	117	120	11	10	134	101	220	233	19	14
	sd	8	7	4	0	10	7	23	16	6	4
	f(I)	0.92	0.80	0.73	0.71	1.24	0.79	0.87	0.97	0.90	0.67
625 µg/pl.	Mean	98	127	13	13	146	97	209	195	20	21
	sd	3	30	1	2	5	5	24	42	2	2
	f(I)	0.77	0.85	0.87	0.93	1.35	0.76	0.83	0.81	0.95	1.00
313 µg/pl.	Mean	123	113	14	14	141	96	240	195	20	20
	sd	15	19	1	1	23	18	28	27	1	1
	f(I)	0.97	0.75	0.93	1.00	1.31	0.75	0.95	0.81	0.95	0.95

f(I) = increase factor

* Different positive controls were used

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test item KCCS DOB11 is considered as “not mutagenic under the conditions of the test”.

Executive summary:

The test item is considered not mutagenic for the reasons given above.

The test item showed a very slight cytotoxicity towards the bacteria strain TA1535 in the cytotoxicity test in the treatment without metabolic activation. This can be seen as uncritical, because the test item showed no cytotoxic effects towards this strain in the main experiment.

The confirmation tests of the genotype didn’t show any irregularities. The control of the titre was above the demanded value. The numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory and were definitely increased in comparison with the negative controls, as well as showing mutagenic potential of the diagnostic mutagenes.

Spontaneous revertants were within the normal range in comparison with the historical data of the LAUS GmbH.

For these reasons, the result of the test is considered valid.