Titanium complexes of ethanol and 2-dimethylaminoethanol

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:

From 2001-01-09 to 2001-03-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

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

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (liver post mitochondrial fraction of rats induced with phenobarbitone/ beta-naphtoflavone)

Test concentrations with justification for top dose:

- First mutagenicity experiment with and without S9 mix: 0, 50, 150, 500, 1500 and 5000 µg/plate
- Second mutagenicity experiment with and without S9 mix: 0, 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

vehicle used: Ethanol

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- In agar (plate incorporation): first mutagenicity and second mutagenicity tests with or without S9 mix
- Exposure duration: 48h

SELECTION AGENT (mutation assays): agar containing traces of histidine and biotin, maintained at 45°C for Salmonella strains.
agar containing traces of tryptophan and biotin, maintained at 45°C for Escherichia coli.

NUMBER OF REPLICATIONS: two independent mutagenicity experiments each using three plates/dose-level

DETERMINATION OF CYTOTOXICITY
- Method: the evaluation of the toxicity was based on the decrease in the number of revertant colonies and/or thinning of the bacterial lawn.

Evaluation criteria:

After incubation for 48 hours at 37 °C in the dark, colonies were counted

Statistics:

No statistical analysis performed

Results and discussion

Test results

Test results 1

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

Remarks:

Tested up to limit concentrations recommended by the test guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to limit concentrations recommended by the test guideline

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

- The number of revertants for the vehicle and positive controls were as specified in the acceptance criteria. The study was therefore considered as valid.

 - Since the test item was freely soluble and non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

- A white precipitate was observed at 5000 µg/plate, this did not prevent the scoring of revertant colonies

- No toxicity was noted at any dose-level in any strain.

- The test item did not induce any noteworthy increase in the number of revertants, in any of the strains, either with or without S9 mix.

Table 7.6.1/1 Experiment 1- Without metabolic activation

With or Without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA 100	TA 1535	WP2uvrA-	TA 98	TA 1537
-	0	102      (91)  86 9.2 86	12      (12)12 0.6 13	39       (28) 25 9.8 20	40       (28) 23 10.8 20	7         (6) 9 3.6 2
-	50	95       (94) 87 6.6 100	13       (13) 13 0.0 I3	25       (29) 30 3.2 31	18       (21) 22 2.6 23	7         (6) 4 2.1 8
-	150	85       (86) 84 2.1 88	13       (13) 12 1.0 14	22       (26) 34 6.9 22	22       (18) 18 4.5 13	3         (3) 3 0.0 3
-	500	92       (93) 97 3.2 91	14       (13) 12 1.2 12	20       (23) 25 2.5 23	18       (19) 19 0.6 19	4         (6) 6 1.5 7
-	1500	92       (87) 79 7.0 90	15      (13) 12 2.1 11	22        (25) 31 4.9 23	16       (21) 24 4.6 24	6         (7) 5         2.1 9
-	5000	88P (95) 89P 11.8 109P	llP (11) 11P 0.0 11P	23P (26) 25P 4.2 31P	24P (33) 38P       38P       8.1	9P       (9) 9P 0.0 9P
Positive controls   S9-Mix -	Name   Concentration (µg/plate)   No.colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		467      (479) 498       16.4 473	249     (240) 213      24.2 259	811     (802) 769      29.5 826	107     (107) 106      0.6 107	1801   (1826) 1649    190.2 2027

Table 7.6.1/2 Experiment 1- With metabolic activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA 100	TA 1535	WP2uvrA-	TA98
+	0	147     (131) 124 123     13.6	17       (16) 14 17       1.7	22       (23) 22 25        1.7	43       (31) 26 25       10.1	6         (7) 6 10       2.3
+	50	125     (123) 120 125      2.9	17       (16) 16 15       1.0	31       (26) 23 25        4.2	35      (29) 30 21        7.1	11       (9) 8 7         2.1
+	150	85       (92) 102 90       8.7	17       (17) 18 17       0.6	23      (27) 33 26        5.1	31      (33) 37 31        3.5	8         (6) 2 9         3.8
+	500	.134     (115) 109 103      16.4	10      (12) 13 12        1.5	30       (29) 31 27        2.1	24       (29) 28 34        5.0	7         (9) 10 Il       2.1
+	1500	112     (89) 96 60      26.6	10       (12) 16 11       3.2	24       (23) 21 25       2.1	27       (29) 29 31       2.0	7         (6) 5 7         1.2
+	5000	l01P     (96) l24P 64P      30.3	16P     (16) 16P 16P     0.0	28P      (26) 22P 27P       3.2	26P      (32) 34P 37P       5.7	5P        (6) 5P 8P        1.7
Positive controls   S9-Mix   +	Narne   Concentration (µg/plate)   No.colonies per plate	2AA	2AA	2AA	BP	2AA
		1	2	10	5	2
		1855   (1740) 1809    160.4 1557	472     (388) 357      73.6 335	965    (1024) 1029    56.2 1077	277     (240) 216      32.5 227	346    (467) 537     105.5 519

Table 7.6.1/3 Experiment 2- Without metabolic activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA 100	TA 1535	WP2uvrA-	TA 98	TA 1537
-	0	157     (146) 133 149     12.2	16      (19) 15 26 6.1	45       (36) 35 28 8.5	45       (41) 39 40 3.5	20       (15) 14       4.2 12
-	50	150    (140) 135 134      9.0	20       (19) 17 20        1.7	48      {37) 33 31       9.3	29      (36) 45 33       8.3	9        (15) 18 17       4.9
-	150	160     (147) 128 154     17.0	24       (21) 15 25        5.5	.31        (32) 29 37        4.2	45       (48) 42 57       7.9	7        (13) 17 16       5.5
-	500	158     (146) 141 140      10.1	16      (18) 14 25       5.9	28      (35) 38 40        6.4	46      (53) 54 58       6.1	17      (19) 20 19        1.5
-	1500	122     (124) 129 122      4.0	30       (24) 15 7.8 26	39    (37) 38 3.2 33	39      (40) 40 40 0.6	20       (19) 18 19 1.0
-	5000	121P  (128) 114P    18.0 148P	19P      (17) 15P 2.1 16P	32P      {36) 41P       4.6 35P	44P      (35) 32P 7.9 29P	12P     (12) 14P 2.5 9P
Positive controls   S9-Mix   -	Name   Concentration (µg/p1ate)   No.colonies per plate	ENNG	ENNG	ENNG	4NQO	9AA
		3	5	2	0.2	80
		663     (680) 697      17.0 681	279     (255) 266      30.4 221	639     (596) 606     49.3 542	109    (108) 113      5.0 103	946     (881) 829     59.7 867

Table 7.6.1/4 Experiment 2- With metabolic activation

With or without S9-Mix	Test substance concentration (µg/plate)	Number of revertants (mean number of colonies per plate)
		TA 100	TA 1535	WP2uvrA-	TA 98	TA 1537
+	0	137     (146) 154 148     8.6	22       (19) 19 17       2.5	42       (44) 43 47        2.6	54       (53) 52        1.4 C	15       (16) 14 20        3.2
+	50	151     (143) 152 127     14.2	17      (26) 31 29        7.6	39       (41) 49 35       7.2	58       (53) 53 49       4.5	17       (15) 10 19       4.7
+	150	148     (144) 148 137      6.4	19       (24) 24 30       5.5	45       (44) 47 39        4.2	57       (57) 57 57       0.0	13       (19) 21 22        4.9
+	500	125     (134) 128 149      13.1	24       (22) 21 21        1.7	52  (38) 34 28       12.5	64      (61) 58 61        3.0	19       (l5) 14 13        3.2
+	1500	119     (130) 132 138      9.7	26       (27) 21 33        6.0	38       (41) 43 41       2.5	55 43 (54) 63       10.1	10      (14) 20 13        .5.1
+	5000	130P    (136) 155P 124P     16.4	24P      (23) 25P      2.1 21P	26P      (32) 30P 41p      7.8	57P      (59) 64P      4.4 56P	llP       (11) l2P llP        0.6
Positive controls   S9-Mix   +	Name   Concentration (µg/plate)   No.colonies per plate	2AA	2AA	2AA	BP	lAA
		1	2	10	5	2
		2202   (2240) 2286     42.6 2232	308     (295) 286      11.4 292	991     (985) 940      42.3 1024	240     (236) 220      14.0 247	570     (560) 552       9.1 559

2AA: 2 -Aminoanthracene

BP: Benzo(a)pyrene

P: Precipitate

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO: 4 -Nitroquinoline-1 -oxide

9AA: 9 -Aminoacridine

C: Contaminated

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-mutagenic under the conditions of this test.

Executive summary:

The potential of the substance to induce reverse mutation in bacteria was assessed using four strains of Salmonella typhimurium and Escherichia Coli WP2 uvrA according to the OECD guideline 471.The study was conducted in compliance with Good Laboratory Practices.

A preliminary toxicity test was performed to define the dose-levels of the test substance to be used for the mutagenicity study (all doses were expressed in pure substance). The test item was then tested in two independent experiments, both with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with phenobarbitone and beta-naphtoflavone.

Both experiments were performed according to the direct plate incorporation. After 48 hours of incubation at 37°C, the revertant colonies were scored. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The preliminary assays with and without metabolic activation showed that the test substance was freely soluble and non-toxic in all tested strains. The highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines with a dose range of 50 to 5000 µg/plate.

In the main experiment, the number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

A white precipitate was observed in the petri plates at 5000 µg/plate, this did not prevent the scoring of the colonies. No reduction in the growth of the bacterial background lawn was observed.

In the two independent assays, no significant increase in the mean number of revertants was noted in the bacterial strains tested in the presence of the test substance neither with nor without metabolic activation.

Under these experimental conditions, the substance did not show any mutagenic activity in the bacterial reverse mutation test.