Amines, C12-14-alkyl, C6-10-alkyl phosphates

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

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:

Amines, C12-14-alkyl, C6-10-alkyl phosphates

EC Number:

271-663-3

EC Name:

Amines, C12-14-alkyl, C6-10-alkyl phosphates

Cas Number:

68603-55-4

Molecular formula:

not available (UVCB)

IUPAC Name:

Amines, C12-14-alkyl, C6-10-alkyl phosphates

Test material form:

liquid: viscous

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 97

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not specified

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not specified

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

not specified

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone. Acetone was chosen as vehicle, because the test item was sufficiently soluble in this solvent, and it does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations. On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in acetone was prepared. The test item solution was not sterile filtrated before use. The stock solution was used to prepare the geometric series of the concentrations to be tested. The following nominal concentrations were prepared for the experiment 1a: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate. On the day of the start of the experiment 1b and 2a, a stock solution containing 5 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the experiment 1b: 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL/plate, 0.005 µL/plate and 0.0015 µL/plate. The following nominal concentrations were prepared for the experiment 2a: 0.5 µL/plate, 0.25 µL/plate, 0.125 µL/plate, 0.063 µL/plate, 0.031 µL/plate, 0.016 µL/plate and 0.08 µL/plate. On the day of the start of the experiment 2b, a stock solution containing 1.25 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the experiment 2b for the bacteria strains TA98, TA102 and TA1535: 0.125 µL/plate, 0.063 µL/plate, 0.031 µL/plate, 0.016 µL/plate, 0.008 µL/plate and 0.004 µL/plate. The following nominal concentrations were prepared for the experiment 2b for the bacte-ria strains TA97a and TA100: 0.016 µL/plate, 0.008 µL/plate, 0.004 µL/plate, 0.002 µL/plate, 0.001 µL/plate and 0.0005 µL/plate.

Vehicle / solvent:

Acetone

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene Diamine, 2-Amino-Anthracene,

Details on test system and experimental conditions:

Mutation present in strain
Name Category Effect TA97a TA98 TA100 TA102 TA1535
hisD6610 frame shift histidine deficiency x
hisD3052 frame shift histidine deficiency x
hisG46 base pair substitution histidine deficiency x x
hisG428 base pair substitution histidine deficiency x
uvrB deletion UV sensitivity, biotin deficiency x x x x
rfa deletion lipopolysaccharide side chain x x x x x
pKM101 plasmid ampicillin resistance x x x x
pAQ1 plasmid tetracycline re-sistance x

All Salmonella typhimurium strains were obtained from TRINOVA BioChem GmbH (batch: TA97a: 4997D, TA98: 5011D, TA100: 4996D, TA102: 4982D, TA1535: 5012D) and were stored as lyophilizates in the refrigerator at 2-8 °C.
The lyophilizates were used to prepare permanent cultures which were filled into vials and stored at < - 75 °C.

Eight hours before the start of each experiment, an aliquot of a permanent culture per strain to be used was taken from the deep freezer to inoculate a culture vessel containing nutrient broth. After incubation overnight for eight hours at 37 ± 1 °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.

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The test item showed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation (details see in other information on results)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Any other information on results incl. tables

The detailed data of all the experiments are listed in the annex (Exp. 1a see chapter 12, page 35ff., Exp. 1b see chapter 13, page40ff., Exp. 2a see chapter14, page 45, Exp. 2b see chapter 15, page 50).

Confirmation of genotype is performedfor each batch of lyophilized bacteria before stock culture preparation. The last performance showed no abnormalities .

1.1     Experiment 1a

1.1.1   Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter 17, page 57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

1.1.2   Solubility and Toxicity

In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.

At the three highest concentrations (5, 1.5 and 0.5 µL/plate) no bacteria growth and no bacterial lawn was observed. The test itemshowed signs of toxicity towards all the bacteria strains in both the absence and presence of metabolic activation in these concentrations.

At the two lower concentrations, the bacterial background lawn was not reduced and no decrease of the spontaneous revertants was observed.

1.1.3   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.

 

Based on the toxicity results, a repetition of this experiment with lower concentrations was performed.

 

1.1.4   Survey of the Findings

The mean revertant values of the three replicates are presented in the following table.

Table8.1‑a      Mean Revertants Experiment 1a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	73	81	26	25	77	93	308	353	19	23
	sd	7.6	11.3	4.7	6.2	7.5	7.0	45.1	4.6	1.0	4.2
DMSO	Mean	67	85	26	29	72	83	305	292	19	21
	sd	3.6	13.0	3.6	6.5	7.8	9.5	45.5	56.7	3.8	4.7
Acetone	Mean	76	83	28	30	80	93	344	329	19	21
	sd	17.2	20.7	4.7	5.6	10.2	11.8	47.2	84.3	3.5	3.0
Positive Controls*	Mean	520	609	407	86	324	1001	1005	1363	224	247
	sd	84.3	136.8	56.0	6.8	55.6	0.0	334.9	224.2	49.2	48.1
	f(I)	7.76	7.16	15.65	2.97	4.21	12.06	3.30	4.67	11.79	11.76
5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
1.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.15 µL/plate	Mean	69	59	20	31	73	82	255	227	22	16
	sd	2.9	7.2	1.0	1.7	7.5	14.4	82.4	44.1	5.0	4.7
	f(I)	0.91	0.71	0.71	1.03	0.91	0.88	0.74	0.69	1.16	0.76
0.05 µL/plate	Mean	74	81	27	29	83	79	277	308	14	18
	sd	10.3	12.0	5.2	4.6	8.0	11.3	73.4	39.4	3.6	1.0
	f(I)	0.97	0.98	0.96	0.97	1.04	0.85	0.81	0.94	0.74	0.86

f(I) = increase factor, calculation see chapter7.4, page23

* Different positive controls were used, see chapter6.3, page13

1001 colonies per plate means the bacteria growth was too strong for counting

 

 

1.2     Experiment 1b

1.2.1   Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

1.2.2   Solubility and Toxicity

In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.

At the highest concentration (0.5 µL/plate), no bacteria growth was observed in the following bacteria strains: TA97a, TA98 and TA100. Towards the bacteria strains TA102 and TA1535 a clear decrease in the spontaneous revertants was observed.

The bacterial background lawn was visible in all concentrations.

1.2.3   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.

 

1.2.4   Survey of the Findings

The mean revertant values of the three replicates are presented in the following table.

Table8.2‑a      Mean Revertants Experiment 1b

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	79	100	32	34	97	98	311	285	12	16
	sd	12.6	17.6	3.1	2.5	5.1	14.2	28.1	78.0	4.4	1.5
DMSO	Mean	89	84	36	31	93	107	290	308	12	18
	sd	15.0	0.6	4.6	1.5	8.5	9.9	67.7	45.1	2.9	1.5
Acetone	Mean	79	81	32	32	99	105	432	363	20	15
	sd	5.7	15.2	4.0	2.3	11.4	6.1	82.7	48.1	7.5	2.9
Positive Controls*	Mean	605	747	608	155	291	1001	1496	817	413	201
	sd	101.7	61.1	89.1	63.6	33.5	0.0	72.0	86.9	177.1	18.5
	f(I)	6.80	8.89	16.89	5.00	3.00	9.36	5.16	2.65	34.42	11.17
0.5 µL/plate	Mean	0	0	0	0	0	0	68	57	3	2
	sd	0.0	0.0	0.0	0.0	0.0	0.0	25.5	4.2	0.6	1.2
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.16	0.16	0.15	0.13
0.15 µL/plate	Mean	67	80	29	30	80	90	241	209	13	12
	sd	8.0	12.5	7.8	2.0	12.8	11.9	68.4	35.9	1.0	3.8
	f(I)	0.85	0.99	0.91	0.94	0.81	0.86	0.56	0.58	0.65	0.80
0.05 µL/plate	Mean	84	82	35	28	72	96	235	311	18	16
	sd	0.6	15.9	1.5	6.2	8.0	6.7	15.1	47.7	2.3	2.9
	f(I)	1.06	1.01	1.09	0.88	0.73	0.91	0.54	0.86	0.90	1.07
0.015 µL/plate	Mean	76	83	25	32	88	110	265	292	14	17
	sd	10.4	11.1	4.7	6.5	6.0	12.0	74.2	6.9	2.5	1.7
	f(I)	0.96	1.02	0.78	1.00	0.89	1.05	0.61	0.80	0.70	1.13
0.005 µL/plate	Mean	100	96	33	32	99	99	249	273	13	14
	sd	22.1	26.5	10.1	3.6	14.7	24.1	12.9	19.7	3.1	2.1
	f(I)	1.27	1.19	1.03	1.00	1.00	0.94	0.58	0.75	0.65	0.93
0.0015 µL/plate	Mean	71	66	35	25	95	101	233	239	13	11
	sd	4.7	11.0	5.2	5.5	11.7	18.0	48.1	38.9	1.7	2.1
	f(I)	0.90	0.81	1.09	0.78	0.96	0.96	0.54	0.66	0.65	0.73

f(I) = increase factor, calculation see chapter7.4, page23

* Different positive controls were used, see chapter6.3, page13

1001 colonies per plate means the bacteria growth was too strong for counting

 

 

1.3      Experiment 2a

1.3.1   Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

1.3.2   Solubility and Toxicity

In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.

At the three highest concentrations (0.5, 0.25 and 0.125 µL/plate) no bacteria growth and no bacterial lawn was visible. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.

Towards the bacteria strains TA97a and TA100, signs of toxicity were also observed in the concentrations 0.063, 0.031 and 0.016 µL/plate.

1.3.3   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.

 

Due to the toxicity results, the experiment was repeated.

1.3.4   Survey of the Findings

The mean revertant values of the three replicates are presented in the following table.

Table8.3‑a    Mean Revertants Experiment 2a

Strain		TA97a		TA98		TA100		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	74	81	36	33	84	100	280	341	21	11
	sd	5.6	5.0	3.1	2.1	11.1	19.1	34.6	124.7	7.0	1.0
DMSO	Mean	76	78	33	35	84	92	283	343	12	12
	sd	16.0	13.6	4.2	3.6	8.5	13.9	14.0	37.2	2.0	2.6
Acetone	Mean	80	85	38	33	83	77	301	349	15	13
	sd	1.0	9.0	15.0	7.2	9.8	5.9	20.1	42.8	3.2	1.7
Positive Controls*	Mean	295	495	129	128	269	361	661	720	272	99
	sd	37.8	177.6	23.4	24.3	83.9	2.3	59.4	14.4	90.1	12.9
	f(I)	3.88	6.35	3.91	3.66	3.20	3.92	2.34	2.10	12.95	8.25
0.5 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.25 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.125 µL/plate	Mean	0	0	0	0	0	0	0	0	0	0
	sd	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
	f(I)	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00
0.063 µL/plate	Mean	2	6	37	29	14	9	280	239	13	14
	sd	1.2	0.0	5.9	7.9	3.8	1.5	28.0	37.0	1.2	3.2
	f(I)	0.03	0.07	0.97	0.88	0.17	0.12	0.93	0.68	0.87	1.08
0.031 µL/plate	Mean	21	32	36	32	23	45	277	297	13	14
	sd	2.1	13.0	7.6	5.0	15.5	3.6	51.4	52.8	0.0	3.5
	f(I)	0.26	0.38	0.95	0.97	0.28	0.58	0.92	0.85	0.87	1.08
0.0016 µL/plate	Mean	10	36	31	32	21	39	264	285	15	17
	sd	1.2	7.2	7.1	7.8	7.2	9.9	52.9	30.6	3.5	2.1
	f(I)	0.13	0.42	0.82	0.97	0.25	0.51	0.88	0.82	1.00	1.31
0.008 µL/plate	Mean	65	83	36	36	82	83	265	248	14	16
	sd	6.0	7.6	4.5	4.2	8.2	9.8	30.6	74.1	3.2	1.7
	f(I)	0.81	0.98	0.95	1.09	0.99	1.08	0.88	0.71	0.93	1.23

f(I) = increase factor, calculation see chapter7.4, page23

* Different positive controls were used, see chapter6.3, page13

 

 

 

1.4     Experiment 2b

1.4.1   Confirmation of the Criteria and Validity

All strains met the criterion of at least 10⁹bacteria/mL, and no inconsistencies were found in the sterility control. All determined values for the spontaneous revertants of the negative controls were in the normal range of the test laboratory (historical data of the laboratory see chapter17, page57).All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.

1.4.2   Solubility and Toxicity

In this experiment, the test itemshowed no precipitates on the plates in all tested concentrations.

Signs of toxicity were observed in the following concentrations towards the respective bacteria strains:

·        TA97a: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA98: 0.125 µL/plate (in the treatment without S9 mix no bacteria growth was observed, in the treatment with S9 mix a decrease in the spontaneous revertants was observed)

·        TA100: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA102: 0.125 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA1535: 0.125 µL/plate (no bacteria growth was observed in the treatment with and without S9 mix)

1.4.3   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.

1.4.4   Survey of the Findings

The mean revertant values of the three replicates are presented in the following table.

Table8.4‑a    Mean Revertants Experiment 2b

Strain		TA98		TA102		TA1535
Induction		-S9	+S9	-S9	+S9	-S9	+S9
Demin. water	Mean	31	35	269	244	26	22
	sd	5.1	4.7	12.2	4.0	4.0	3.5
DMSO	Mean	35	33	307	311	21	21
	sd	3.5	6.9	46.9	6.1	3.5	1.5
Acetone	Mean	35	40	253	264	23	25
	sd	5.5	3.1	10.1	42.3	3.2	3.5
Positive Controls*	Mean	441	104	1320	1312	331	272
	sd	18.9	18.0	36.7	42.3	53.3	32.0
	f(I)	12.60	3.15	4.30	4.22	12.73	12.95
0.125 µL/plate	Mean	0	3	90	58	0	0
	sd	0.0	2.9	24.0	5.3	0.0	0.0
	f(I)	0.00	0.08	0.36	0.22	0.00	0.00
0.063 µL/plate	Mean	28	31	264	223	20	20
	sd	4.4	6.0	0.0	20.1	6.0	2.5
	f(I)	0.80	0.78	1.04	0.84	0.87	0.80
0.031 µL/plate	Mean	28	34	240	277	20	20
	sd	8.9	6.0	22.3	55.5	3.2	2.6
	f(I)	0.80	0.85	0.95	1.05	0.87	0.80
0.016 µL/plate	Mean	37	33	225	252	19	19
	sd	3.1	3.5	34.0	34.9	5.7	3.6
	f(I)	1.06	0.83	0.89	0.95	0.83	0.76
0.008 µL/plate	Mean	34	32	244	265	21	17
	sd	2.1	4.6	4.0	18.5	4.4	0.6
	f(I)	0.97	0.80	0.96	1.00	0.91	0.68
0.004 µL/plate	Mean	35	32	223	264	18	22
	sd	2.1	5.1	18.9	28.0	2.6	3.1
	f(I)	1.00	0.80	0.88	1.00	0.78	0.88

f(I) = increase factor, calculation see chapter7.4, page23

* Different positive controls were used, see chapter6.3, page13

1001 colonies per plate means the bacteria growth was too strong for counting

 

Table8.4‑b    Mean Revertants Experiment 2b

Strain		TA97a		TA100
Induction		-S9	+S9	-S9	+S9
Demin. water	Mean	83	81	115	116
	sd	14.4	8.1	7.5	8.4
DMSO	Mean	76	90	86	92
	sd	11.6	10.6	8.4	12.5
Acetone	Mean	85	78	93	105
	sd	7.2	14.2	12.2	14.3
Positive Controls*	Mean	331	313	531	1001
	sd	40.1	34.0	45.5	0.0
	f(I)	4.36	3.48	4.62	10.88
0.016 µL/plate	Mean	16	23	33	31
	sd	0.0	7.4	3.8	5.5
	f(I)	0.19	0.29	0.35	0.30
0.008 µL/plate	Mean	65	85	79	96
	sd	1.2	21.9	4.6	25.5
	f(I)	0.76	1.09	0.85	0.91
0.004 µL/plate	Mean	75	80	93	114
	sd	16.7	5.3	15.0	11.1
	f(I)	0.88	1.03	1.00	1.09
0.002µL/plate	Mean	76	79	81	107
	sd	8.4	11.4	5.5	9.5
	f(I)	0.89	1.01	0.87	1.02
0.001 µL/plate	Mean	71	100	83	110
	sd	14.7	16.8	6.4	17.9
	f(I)	0.84	1.28	0.89	1.05
0.0005µL/plate	Mean	74	106	102	114
	sd	7.2	15.9	22.8	8.7
	f(I)	0.87	1.36	1.10	1.09

f(I) = increase factor, calculation see chapter7.4, page23

* Different positive controls were used, see chapter6.3, page13

1001 colonies per plate means the bacteria growth was too strong for counting

 

 

 

1.5     Mutagenicity of Test Item

The test item Deophos 228showed no increase in the number of revertants in all bacteria strains in all experiments.

All negative and all strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded thatDeophos 228is not mutagenic in theSalmonella typhimuriumtest strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in the present study.

Applicant's summary and conclusion

Conclusions:

In all experiments, no precipitation of the test item Deophos 228 was observed at any of the tested concentrations up to 5 µL/plate. The test item showed toxicity towards all bacteria strains in all experiments. The confirmation tests of the genotype did not show any irregularities. The control of the titre was above the demanded value of 109 bacteria/mL. All of the means of all replicates of the spontaneous revertants (in negative and solvent controls) were within the range of the historical data of the test facility. All numbers of revertant colonies of the positive controls were within the range of the historical data of the laboratory (historical data of the laboratory chapter 17, page 57) and were increased in comparison with the negative controls, which demonstrated the mutagenic potential of the diagnostic mutagens. Since all criteria for acceptability have been met, the study is considered valid.

Executive summary:

Four valid experiments were performed. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Deophos 228 was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in four experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.  

Experiment 1a:

In this experiment,the test item (dissolved in acetone) was tested up to concentrations of 5  µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the three highest concentrations (5, 1.5 and 0.5 µL/plate) no bacteria growth and no bacterial lawn was observed. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.

At the lower two concentrations, the bacterial background lawn was not reduced and no decrease of the spontaneous revertants was observed.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 1b:

Based on the toxicity results of the experiment 1a,the test item was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in all bacteria strains using the plate incorporation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the highest concentration (0.5 µL/plate), no bacteria growth was observed in the following bacteria strains: TA97a, TA98 and TA100. Towards the bacteria strains TA102 and TA1535 a clear decrease in the spontaneous revertants was observed.

The bacterial background lawn was observed in all concentrations.

 

The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

 

Experiment 2a:

In this experiment, the test item (dissolved in acetone) was tested up to concentrations of 0.5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the pre-incubation method.

The test item showed no precipitates on the plates at any of the concentrations.

At the three highest concentrations (0.5, 0.25 and 0.125 µL/plate) no bacteria growth and no bacterial lawn was visible. The test itemshowed signs of toxicity towards all bacteria strains in both the absence and presence of metabolic activation in these concentrations.

Towards the bacteria strains TA97a and TA100, signs of toxicity were observed in the concentrations 0.063, 0.031 and 0.016 µL/plate, too.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

 

Experiment 2b:

In this experiment, the test item was tested up to concentrations of 0.125 µL/plate in the absence and presence of S9-mix in the bacteria strains TA98, TA102 and TA1535 resp. up to concentrations of 0.016 µL/plate in the absence and presence of S9-mix in the bacteria strains TA97a and TA100.

The test item showed no precipitates on the plates at any of the concentrations.

Signs of toxicity were observed in the following concentrations towards the respective bacteria strains:

·        TA97a: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA98: 0.125 µL/plate (in the treatment without S9 mix no bacteria growth was observed, in the treatment with S9 mix a decrease in the spontaneous revertants was observed)

·        TA100: 0.016 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA102: 0.125 µL/plate (a decrease in the spontaneous revertants with and without S9 mix)

·        TA1535: 0.125 µL/plate (no bacteria growth was observed in the treatment with and without S9 mix)

 

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Based on the results of this study it is concluded that Deophos 228 is not mutagenic in the Salmonella typhimuriumstrains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.