Everzol Red CDN Crude

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From January 25, 2008 to March 17, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

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:

rat or hamster liver S9-mix

Controls

Negative solvent / vehicle controls:

yes

Remarks:

Milli-Q water

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

congo red

methylmethanesulfonate

other: Sodium azide, 2-aminoanthracene

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Remarks:

hamster liver S9-mix.

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Experiment 1 was a direct plate assay with rat liver S9-mix.

Experiment 2 was a preincubation assay with hamster liver S9-mix.

Experiment 3 was performed with tester strain TA100 in the presence of hamster liver S9-mix.

Table 1.Experiment 1: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	881 ± 15	356 ± 97	1002 ± 45	1007 ± 16	1288 ± 76
Solvent control	15 ± 4	4 ± 1	14 ± 3	115 ± 4	24 ± 6
3				95 ± 9	25 ± 4
10				108 ± 19	25 ± 1
33				105 ± 6	20 ± 5
100	9 ± 1	5 ± 1	18 ± 5	111 ± 13	28 ± 11
333	13 ± 3	5 ± 1	20 ± 6	115 ± 11	25 ± 4
1000	11 ± 1	5 ± 2	15 ± 1	109 ± 5	25 ± 5
3330	10 ± 1	3 ± 1	18 ± 3	96 ± 6	35 ± 3
5000	10 ± 1	2 ± 1	18 ± 5	102 ± 7	32 ± 4
With S9-mix
Positive control	220 ± 17	431 ± 32	891 ± 58	886 ± 60	420 ± 36
Solvent control	9 ± 6	5 ± 1	16 ± 2	81 ± 3	30 ± 5
3				73 ± 6	32 ± 6
10				78 ± 10	22 ± 6
33				91 ± 6	34 ± 4
100	12 ± 6	4 ± 1	18 ± 3	102 ± 10	28 ± 8
333	11 ± 2	3 ± 1	15 ± 2	105 ± 16	34 ± 2
1000	13 ± 1	4 ± 2	17 ± 2	113 ± 11	33 ± 7
3330	12 ± 4	3 ± 1	21 ± 1	91 ± 6	35 ± 10
5000	13 ± 3	4 ± 2	18 ± 4	109 ± 7	36 ± 3

Table 2. Experiment 2: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and Escherichia coli strain
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-mix
Positive control	847 ± 31	185 ± 37	975 ± 41	675 ± 59	240 ± 24
Solvent control	10 ± 3	3 ± 1	17 ± 4	112 ± 6	25 ± 9
100	7 ± 3	3 ± 1	19 ± 2	114 ± 9	22 ± 5
333	19 ± 6	4 ± 1	18 ± 2	97 ± 2	25 ± 4
1000	11 ± 1	4 ± 1	20 ± 2	106 ± 20	20 ± 3
3330	9 ± 5	4 ± 2	21 ± 3	104 ± 11	20 ± 2
5000	12 ± 2	3 ± 1	20 ± 2	85 ± 2	27 ± 2
With S9-mix
Positive control	185 ± 37	187 ± 23	1468 ± 55	833 ± 314	323 ± 41
Positive control (CR)			381 ± 20
Solvent control	6 ± 2	5 ± 1	29 ± 5	71 ± 12	28 ± 2
100	5 ± 2	7 ± 3	33 ± 7	66 ± 5	30 ± 1
333	10 ± 2	10 ± 4	35 ± 5	68 ± 4	32 ± 2
1000	6 ± 3	8 ± 4	36 ± 4	80 ± 5	29 ± 4
3330	12 ± 2	7 ± 0	22 ± 7	153 ± 10	27 ± 4
5000	8 ± 3	7 ± 4	24 ± 2	173 ± 17	31 ± 6

CR: Congo red

 

Table 3. Experiment 3: Mutagenic response of Everzol Red Crude in the Salmonella typhimurium reverse mutation assay

Dose (μg/plate)	Mean number of revertant colonies/3 replicate plates (±S.D.) one strains of Salmonella typhimurium
	TA100
With S9-mix
Positive control	505 ± 20
Solvent control	80 ± 4
100	84 ± 10
333	90 ± 12
1000	95 ± 5
3330	161 ± 12
5000	194 ± 32

Applicant's summary and conclusion

Conclusions:

According to OECD 471 test method and EU Method B.13/14, Everzol Red CDN Crude is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix in tester stain TA100.

Executive summary:

This test using the procedures outlined in the NOTOX Study Plan for 487480, OECD 471 (OECD, 1997) and EU Method B.13/14 (2000). The results of this test for Everzol Red CDN Crude show that test validity criteria was met.

The test was performed in two independent experiments, at first a direct plate assay was performed with rat liver S9-mix and secondly a preincubation assay was performed with hamster liver S9-mix. To obtain more information about the possible mutagenicity of Everzol Red CDN Crude, an additional experiment was performed with tester strain TA100 in presence of hamster liver S9-mix. Based on thedirect plate assay of the strains TA100 and WP₂uvrA, 5000μg/plate was set as the highest dose in this study. In all assay, five doses of Everzol Red CDN Crude at 100, 333, 1000, 3330 and 5000 μg/plate, solvent control and strain-specific positive controls were tested in tester strains TA98, TA100, TA1535, TA1537 and WP₂uvrA in triplicate with or without S9 Mix activation (rat or hamster liver S9-mix). No toxicity was observed in all five tester strains up to 5000μg/plate in the absence and presence of metabolite activations. Results showed that Everzol Red CDN Crude induced an up to 2.4-fold, dose-related increase in the number of revertant colonies compared to the solvent control in tester strain TA100.

Based on the results of this study it is concluded that Everzol Red CDN Crude is mutagenic in the Salmonella typhimurium reverse mutation assay and not mutagenic in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix in tester stain TA100.