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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
Three in vitro mutagenicity tests all showed positive responses. From the QSAR data, we cannot find similar structure in order to judge the mutagenicity. Therefore, in vivo mutagenicity test is conducted.
Link to relevant study records

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Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
The post-mitochondrial fraction (S9) of liver from Aroclor 1254 induced Sprague-Dawley rats was purchased from MOLTOX, Molecular Toxicology Inc., USA and was used for metabolic activation.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 0, 50, 150, 500, 1500, 5000 µg/ml
Concentration range in the main test (without metabolic activation): 0, 50, 150, 500, 1500, 5000 µg/ml
Vehicle / solvent:
Culture medium
Untreated negative controls:
yes
Remarks:
10% sterile deionized water in culture medium was used as the negative control.
Positive controls:
yes
Remarks:
Mitomycin C at 1 µM ( µg/mL) was used as the positive control for 3-hour and 20-hour treatments without S9 activation. Cyclophosphamide at 40 uM was used for 3-hr treatment with S9 activation.
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours
Exposure period (without metabolic activation): 20 hours

Fixation time:
With/without S9: 3h exposure, 24h fixation (experiment 1)

Without S9: 24h exposure, 24h fixation time (experiment 2)

Without S9: 48h exposure, 48h fixation time (experiment 2)

With 3h exposure, 48h fixation (experiment 2)
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
3hr, at the concentraion: 150 µg/ml, 500 µg/ml
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Concurrent Cytotoxicity Test:
Measurements of cytotoxicity for all treated and negative control cultures were conducted at the time of mitotic cell harvest. The cytotoxicity at all concentrations for all three schemes did not show more than 50% cqtotoxicity. The maximum top concentration recommended was used for all three schemes.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Concurrent Cytotoxicity analysis of Everzol Orange ED-G Crude in Chinese Hamster Ovary Cells

 Concentration (μg/mL)  Cell number (X E5 cells) Survival (%)  Cytotoxicity (%) 
Scheme I (-S9, 3h)       
 Negative control 30.7 100.0 0.0 
 50 26.4  86.0  14.0 
 150 30.1 98.0  2.0 
 500 24.7  80.5  19.5 
 1500 24.7  80.5  19.5 
 5000 23.2  75.6  24.4 
Scheme II (+S9, 3h)       
 Negative contral 23.9  100.0  0.0 
50  30.7  100.0  0.0 
150  28.9 100.0  0.0 
500  13.4  56.1 43.9 
1500  13.2  55.2  44.8
5000  26.9  100.0  0.0 
Scheme III (-S9, 20h)       
Negative control 31.2  100.0  0.0 
50 31.6  100.0  0.0 
150  29.2  93.6  6.4 
500  25.5  81.7  18.3 
1500 22.6  72.4  27.6 
5000  19.0  60.9  39.1 
       

Summary of Chromosome aberrations in Chinese Hamster Ovary cells

 Concentration (μg/mL) Aberrant Cells (%)
 Scheme I (-S9, 3h)  
 Negative control, 0
 50 0.5 
 150
 500 1.5 
 1500 0.5 
 5000 0.5 
 Positive control (MMC) 0.33 25 
 Scheme II (+S9, 3h)  
 Negative control, 0
50  1.5 
 150
 500 9.5 
 1500 Too few metaphases 
 5000 Too few metaphases 
 Positive control (CPP) 22.5 
 Scheme III (-S9, 20h)  
 Negative control, 0  0.5
 50  0
 150 0.5 
 500
 1500 Too few metaphases 
5000  Too few metaphases 
   
Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

In conclusion, Everzol Orange ED-G Crude did not induce structural chromosome aberration in CHO cells for all concentrations in the absence of S9 but induce structural chromosome aberration at the concentrations of 150 and 500 µg/ml in the presence of S9 for 3-hour treatment.
Executive summary:

The chromosome aberration assay was conducted in three test schemes: 3-hour exposure both with and without S9 activation (schemes I and II) and 20-hour continuous exposure without S9 (scheme III). Results were conducted in duplicate cultures and with negative control and positive controls concurrently. 100 metaphases for each culture and 200 metaphases for each treatment were scored. Results showed that percents of aberrant cells induced by negative control in the scheme I, II and III were 1%, 0% and 0.5%. The positive control induced significant increases in percents of aberrant cells over the corresponding negative control. Everzol Orange ED-G did not significantly increase the frequencies of structural chromosome aberration in the schemes I and III. However, the frequencies of structural chromosome aberration in the scheme II, at concentration of 150 and 500 um/mL in the 3 hr incubation have significantly increased.

In conclusion, Everzol Orange ED-G crude induced structural chromosome aberration at the concentration of 150 and 500 μg/mL at the presence of S9 for 3-hr treatment. The concentration-response analysis and trend probability were conducted in the 3-hr treatment with S9 and results showed lacking in concentration response.

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
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-fraction and hamster lever S9 mix-fraction
Test concentrations with justification for top dose:
Concentration range in the main test (with and without metabolic activation): 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Vehicle / solvent:
Solvent: Milli-Q water
Untreated negative controls:
yes
Remarks:
the vehicle of the test article, being Milli-Qwater
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
methylmethanesulfonate
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with
Genotoxicity:
positive
Untreated negative controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Untreated negative controls validity:
valid
Additional information on results:
Observations:
First experiment (direct plate assay): in the presence and absence of hamster liver S9-mix

No increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.


Second experiment (pre-incubation assay): in the presence and absence of hamster liver S9-mix

In tester strain TA98, the test substance induced an up to 3.4-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix.
In tester strain TA100, the test substance induced an up to 2.9-fold dose related increase in the number of revertant colonies compared to the solvent control in the resence of S9-mix.
In tester strain WP2uvrA, the test substance induced an up to 3.3-fold dose related increase in the number of revertant colonies compared to the solvent control in the presence of S9-mix.
In the tester strains TA1535 and TA1537, the test substance induced just 2-fold increases in the number of revertant colonies compared to the solvent control in the presence of S9-mix.


Third experiment (pre-incubation assay): in the presence of hamster liver S9-mix

In tester strain TA98, the test substance induced an up to 2.6-fold dose related increase in the number of revertant colonies compared to the solvent control.
In tester strain TA100, the test substance induced an up to 2.3-fold dose related increase in the number of revertant colonies compared to the solvent control.
In tester strain WP2uvrA, the test substance induced an up to 1.7-fold dose related increase in the number of revertant colonies compared to the solvent control.

Based on the bacteria mutagenicity test, Everzol Orange ED-G crude is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: direct plate assay

Experiment 2: mutagenic response of Everzol Orange ED-G Crude in the reverse mutation assay, preincubation system

 Dose (µg/plate) TA1535  TA1537  TA98  TA100  WP2uvrA 
 Without S9 -mix    
 Positive control 881±33 150± 1097± 100 807± 4 278±201 
 Negative control 12±5 6± 33±5 110 ±12 19 ±5
 100 9±2 3±2 27± 106 ± 24 ±
 333 11±4 5±2  21 ±1  106 ±9  19 ±5
1000  10 ±1  4 ±2  26 ±5 121 ±7 21 ±
 3330  11 ±1 ±1 35 ±3 109 ±10  22 ±5
 5000  9 ±4  4 ±1 34 ±3  102 ±13 32 ±
 With S9 -mix          
 Positive control 194 ± 44  133 ± 912 ± 134  833 ± 314  275 ± 23 
 Negative control ± ± 41 ± 71 ± 12  20 ±
 100  7 ±2 ± 117 ± 30  63 ± 23 ±
 333 ± 12 ± 139 ± 71 ± 11  31 ±
 1000 ± 10 ± 140 ± 47  87 ± 19  33 ±
 3330  13 ±1  14 ±1 71 ±9 174 ± 20  51 ±
 5000  10 ±3 12 ± 50 ± 11  204 ± 65 ±

Experiment 3: mutagenic response of Everzol Orange ED-G in the reverse mutation assay, preincubation system

 Dose (µg/plate) TA98  TA100  WP2uvrA 
 Positive control  1388 ±61 505 ± 20  298 ±
 Negative control 47 ± 80 ± 33 ±
 100  77 ±13 78 ± 29 ±
 333  122 ±22 90 ±13 29 ±
 1000 123 ± 105 ± 39 ±
 3330 95 ± 27  187 ± 11  48 ±
 5000 66 ± 179 ± 55 ±
Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

The test substance is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix.
Executive summary:

Evaluation of the mutagenic activity of Everzol Orange ED-G Crude in the Salmonella typhimurium reverse mutation and the Escherichia coli reverse mutation assay (with independent repeat).

 

Everzol Orange ED-G was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in the Escherischia coli reverse mutation assay with a tryptophan-requiring strain of Escherrichia coli (WP2urA). The test was performed in two independent experiments, at first a direct plate assay was performed with rat liver S9-mix (rat liver S9-mix induced by a combination of Phenobarbital and β-naphthoflavone) and secondly a preincubation assay was performed with hamster liver S9-mix (uninduced male Golden Syrian Hamster liver S9-mix). To obtain more information about the possible mutagenicity of Everzol Orange ED-G Crude, an additional experiment was performed with the strains TA98, TA100 and WP2uvrA in presence of hamster liver S9-mix.

 

In the direct plate assay, Everzol Orange ED-G was initially tested in a dose range finding study up to concentrations of 5000 µg/plate in the strains TA100 and WP2urA. Everzol Orange ED-G Crude did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

 

In the preincubation assay, Everzol Orange ED-G was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed, with the exception of tester strain TA98 in the presence of S9-mix.

 

To verify the results obtained in the preincubation assay, an additional experiment was performed with the strains TA98, TA100 and WP2uvrA in presence of hamster liver S9-mix. Everzol Orange ED-G Crude was tested up to concentration of 5000 µg/plate in these tester strains. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed, with the exception of tester strain TA98 in the presence of S9-mix.

 

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

 

In the absence of S9-mix, all bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant dose-related increase in the number of revertants both in the direct plate and preincubation assay.

 

In the presence of hamster S9-mix in the first preincubation test, Everzol Orange ED-G Crude induced up to 2.9 to 3.4-fold, dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA98, TA100 and WP2uvrA. Verification of these results was performed in an additional experiment, in which, Everzol Orange ED-G Crude induced up to 1.7 to 2.6-fold, dose-related increases in the number of revertant colonies compared to the solvent control in the tester strains TA98, TA100 and WP2uvrA. Since the increases in the number of revertant colonies were observed in two independent repeat experiments, the observed increases were outside our historical control data range (except TA100, additional test) and were dose-related, these increases are considered biologically relevant and Everzol Orange ED-G Crude is considered mutagenic.

 

In all tester strains in the presence of rat-S9-mix in the direct plate test and in the tester strains TA1535 and TA1537 in the presence of hamster S9-mix in the preincubation test, Everzol Orange ED-G Crude did not induce a dose-related, two or three-dold, increase in the number of revertant colonies.

 

Based on the results of this study it is concluded that Everzol Orange ED-G Crude is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. The mutagenicity was confined only to incubations with metabolic activation of hamster liver S9-mix.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
L5178Y/TK+/-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate of Sprague-Dawley rats
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 30, 50, 100, 300, 500, 1000, 3000, 5000 µg/ml
Concentration range in the main test (without metabolic activation): 30, 50, 100, 300, 500, 1000, 3000, 5000 µg/ml
Vehicle / solvent:
Culture medium
Untreated negative controls:
yes
Remarks:
Sterile deionized water
Positive controls:
yes
Remarks:
Ethyl methantsulphonate at 320 µM ( µg/mL) was used as the positive control for 4-hour treatments without S9 activation. 2-acetylaminofluorine at 200 µM ( µg/mL) was used as the positive control for 4-hour treatments with S9 activation.
Positive control substance:
2-acetylaminofluorene
ethylmethanesulphonate
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 4 hours
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
not 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'.

Table 1. Viability of L5178Y after 4 Hours Treatment with Everzol Orange ED-G Crude (Mean ± SD, n=3)
Treatment Viability(%)  
Without S9 activation system With S9 activation system  
N.C.a 89.2±3.2 93.5±3.9
30μg/mL 90.4±4.2 93.3±3.0
50μg/mL 90.5±3.6 91.3±1.8
100μg/mL 90.9±2.0 92.8±1.2
300μg/mL 89.8±1.5 86.2±5.1
500μg/mL 91.6±2.1 91.1±2.4
1000μg/mL 90.2±1.9 92.9±1.3
3000μg/mL 80.8±3.3 92.6±1.9
5000μg/mL 84.9±3.7 87.1±2.5
P.C. 90.3±1.8b 87.5±2.3c
       *Viability (%) = alive cell numbers/(alive + dead cell numbers) × 100
       a: Negative control: sterile deionized water
       b: Positive control in non-activation system: 320μg/mL ethylmethanesulfonate (EMS)
       c: Positive control in S9 activation system: 200μg/mL 2-acetylaminofluorine (2AAF)

Table 2. Relative total growth (RTG) of Orange ED-G Crude treated Cells

  Without S9 activation system      With S9 activation system     
Treatment SG  GSG (%)  RTG (%)  SG GSG (%)  RTG (%) 
 N.C. 20.68  100.00  100.00  18.71  100.00  100.00 
30  μg/mL   20.68  100.00  102.22  20.70  110.64  100.10 
50  μg/mL   18.13  87.67  80.19  19.80  105.83  115.95 
100 μg/mL   19.74  95.45  102.66  17.16  91.72  86.17 
300 μg/mL   19.07  92.21 88.70  14.19  75.84  77.61 
500 μg/mL   15.75  76.16 82.20 14.85  79.37  82.30 
1000 μg/mL 12.35  59.72  48.37  8.25  44.09  39.32 
3000 μg/mL   2.70 13.06  5.05  2.00  10.69  4.24 
5000 μg/mL   1.20  5.80  0.69 1.00  5.34  0.34 
P.C.  22.08  106.77  92.85  2.30  12.29  5.18 

SG: suspension growth = [cell density of 24 hours after treatment/ cell density of 0 hour after treatment] X [cell density of 48 hours after treatment/ cell density of 24 hours after treatement]

RSG: Relative suspension growth = SG tes/ SG negative

Table 3. Mutant Frequency of Everzol Orange ED-G Crude Treated Cells after 12 -Day selection period

   P.E.% M.F. (1 E-6) of total colonies  M.F. (1 E-6) of large colonies   M.F. (1 E-6) of small colonies
Without S9 activation system            
 N.C.  94.02

± 1.68

 28.30

± 12.27

14.02

± 7.00

13.95

± 6.89

 30  96.11

± 4.68

33.18

± 22.42

20.92

±

7.95 
11.78

±

15.16 
50  86.00

±

6.42 
66.26

±

24.10 
36.25

±

18.81 
28.45

±

5.34 
100  101.12

±

13.62 
49.80

±

37.81 
13.71

±

15.03 
35.04

±

22.94 
 300 90.44

± 6.58

 87.21±46.83 31.16±31.39   53.24± 18.49 
 500 101.48± 6.61   40.38±13.77 29.22±15.51   10.68± 3.31 
 1000 76.16± 16.31  94.35± 24.60  37.42± 15.80  54.33± 29.99 
 3000 36.39± 2.95  364.93± 18.61  109.61± 41.81  239.28± 56.65 
 5000 11.14± 0.90  277.38± 46.56  139.60± 41.70  134.64± 78.91 
 P.C. 81.76± 4.82  398.63± 14.95  256.74± 16.17  95.68± 20.83 
 With S9 activation system            
 N.C. 97.12±  4.65 39.10± 2.50  11.32± 4.07  27.31± 6.34 
 30 87.86± 7.95  58.80± 17.46  29.92± 14.38  27.61± 2.40 
 50 106.40± 10.63  59.46± 22.93  28.42± 18.54  29.61± 13.39 
 100 91.24± 4.24  49.00± 15.88  16.86± 4.11  31.32± 14.31 
300  99.39± 7.62  50.60± 26.71  24.49± 3.56  56.02± 22.23 
 500 100.70 ±10.22  58.82±9.95 31.30± 11.85  26.38± 20.70 
 1000 86.60± 12.68  98.95± 10.60  28.28± 4.60  67.80± 7.75 
 3000 38.53± 6.22  189.33± 46.84  19.28± 21.08  168.10± 31.65 
 5000 6.21± 3.27  211.24± 103.15  0.00± 0.00  211.24± 103.15 
 P.C. 40.93± 5.59  451.97± 88.06  91.24± 21.87  336.01± 68.47 

Plating efficiency (P.E.%) = (-ln P(0)/ number of cells per well) X 100%, for cells treated without TFT

Mutant frequency (M.F.) = P.E.% in selective medium/ P.E.% in non-selective medium

Conclusions:
Interpretation of results (migrated information):
positive

In summary, the M.F. of L5178Y/TK+/- cells treated with test article "Everzol Orange ED-G Crude" showed positive with a concentration-response relation in the absence and presence of metabolic system. Therefore, under these test conditions, the test article "Everzol Orange ED-G Crude" is mutagenic in the In Vitro Mammalian Cell Gene Mutation Test.
Executive summary:

Everzol Orange ED-G crude is tested in vitro assay to induce the mutation at the thymidine kinase locus of L5178Y mouse lymphoma cells with or without an exogenous metabolic activation system.

 

8 concentrations (30, 50, 100, 300, 500, 1000, 3000 and 5000 μg/mL) of Everzol Orange ED-G, positive (P.C.) and negative control (N.C.) groups were tested in this assay.

 

For cytotoxicity evaluation, the relative suspension growth (RTG) range was covered the range suggested by OECD. In the non-activation system, the RTG was range from 0.69 % to 102.66 %. At the dose of 1000, 3000 and 5000 μg/mL, the RTG values were half of the NC. In the S9 activation system, the RTG was ranged from 0.34 % to 115.95%. At the doses of 1000, 3000 and 5000 μg/mL, the RTG values were half of the N.C. Therefore, the cytotoxicity of Everzol Orange ED-G is positive.

 

In the non-activation system, the mutation frequency (MF) of positive control was over 5 fold than the MF of the negative. The M.F. of 30, 100 and 500 μg/mL was less than 2 fold of the MF of the NC The MF of the other doses was 2 fold over than the MF of the NC These are considered positive response. The concentration-response analysis and trend probability were conducted and the result is positive.

 

In the S9 activation system, the MF of lower doses (30 to 500 μg/mL) was less than 2 fold of the MF of NC and considered negative response. The MF of 1000 and 5000 μg/mL was 2 fold over the MF of NC and considered positive response. The concentration-response analysis and trend probability were conducted and results show a positive.

 

In summary, the MF of L5178Y/ TK cells treated with Everzol Orange ED-G Crude showed positive with a concentration-response relation in the absence and presence of metabolic system. Therefore, under these test conditions, Everzol Orange ED-G Crude is mutagenic in the in vitro Mammalian Cell Gene Mutation Test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Everzol Orange ED-G Crude is not clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of 2000 mg/kg kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

 

Moreover, Everzol Orange ED-G Crude does not provoke DNA damage in the Comet assay in liver and stomach cells under the experimental conditions described in this report.

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study is planned to be conducted in December 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The micronucleus test (OECD 474) is suitable to investigate other potential mechanisms of genotoxicity such as clastogenicity and aneuploidy.
The comet assay (OECD 489) could exam tissues at the site of contact. The substance does not need to pass through many organs before reaching targeted organ. In the view of optimal use of animals, ECHA agreed to perform a comet assay combined with a micronucleus test.
GLP compliance:
yes
Type of assay:
mammalian comet assay
Species:
rat
Strain:
Wistar
Remarks:
Crl:WI (Han)
Sex:
male
Details on test animals and environmental conditions:
Rats: Crl:WI(Han) (outbred, SPF-Quality) are used as test system. These rats are recommended by international guidelines (e.g. OECD and EC). The animals are provided by Charles River, Sulzfeld, Germany.

Young adult animals were selected (6-7 weeks old at the start of treatment). The total number of animals used in the dose range finding study was 3 and in the main study 30. In the Comet main study, 5 male rats were treated in each Everzol Orange ED-G Crude and the vehicle treatment group.

The positive controls EMS and CP were tested in 5 male animals.
The acclimatisation period was at least 5 days before the start of treatment under laboratory conditions. The body weights of the rats at the start of the treatment with Everzol Orange ED-G Crude were within 20% of the sex mean. The mean body weights were 148 ± 6.2 g and the range 137 – 159 g

The mean body weights were 145 ± 7.8 g (range 130 – 165 g) at the start of treatment with the positive control EMS. The rats were identified by a unique number on the tail written with a marker pen. The animals were allocated at random to the treatment groups.

On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health.
Route of administration:
oral: gavage
Vehicle:
Everzol Orange ED-G Crude was suspended in Milli-Ro water. The specific gravity of Milli-Ro water is 1.0 g/mL. Everzol Orange ED-G Crude concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Everzol Orange ED-G Crude concentrations were dosed within 4 hours after preparation.
Details on exposure:
The rats were dosed for three consecutive days (twice daily) with the test item and vehicle by oral gavage (oral intubation with a plastic gavage needle). The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. The rats were dosed twice with the positive control EMS and once with CP.

The first dose of the test item and vehicle was administered at t=0 h. The second and third dose were administered at approximately t=24 h (± 2 h) and t=45 h (± 2 h), respectively. The positive control CP was administered once at t = 0 h and EMS was administered at t=24 (± 2 h) and t=45 (± 2 h). The animals were sacrificed at t = 48 h. The doses were split into two consecutive doses with a 2-3-hour interval for the highest dose group and negative control dose group.

The dosing volume was 10 mL/kg body weight.
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
Control animals:
yes, concurrent vehicle
yes, historical
no
Positive control(s):
The positive control for the micronucleus test was Cyclophosphamide (CP; CAS no. 50-18-0; Baxter B.V., Utrecht, The Netherlands) at 20 mg/kg body weight dissolved in physiological saline. The stock solutions of CP were stored in aliquots at ≤-15°C in the dark and one sample was thawed immediately before use. The route of administration was consistent with those of the test item. The dosing volume was 10 mL/kg body weight.

The positive control for the Alkaline Comet test was Ethyl Methanesulfonate (EMS; CAS no. 62-50-0; Sigma Aldrich, Steinheim, Germany) at 200 mg/kg body weight dissolved in physiological saline. EMS was used within 3 hours after preparation and the route of administration was oral. The dosing volume was 10 mL/kg body weight.
Details of tissue and slide preparation:
Preparation of bone marrow smears for the micronucleus test
- Preparation of bone marrow smears
The supernatant was removed with a Pasteur pipette. Approximately 500 µl serum was left on the
pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell
suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of
96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides
were marked with the study identification number and the animal number. The drop was spread by
moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the
drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol
(Merck) and air-dried overnight. Two slides were prepared per animal.

- Staining of the bone marrow smears
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide
stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were
automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex
(Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V.,
Rijswijk, The Netherlands)

- Analysis of the bone marrow smears for micronuclei
To prevent bias, all slides were randomly coded before examination. An adhesive label with study
identification number and code was stuck over the marked slide. At first the slides were screened at a
magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread,
undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 4000 polychromatic erythrocytes
(with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was
determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were
calculated. Parts on the slides that contained mast cells that might interfere with the scoring of
micronucleated polychromatic erythrocytes were not used for scoring.


Preparation of Comet slides
- Determination of cell viability
The viability of the cells after isolation was determined by manually counting the number of viable cells
using trypane blue staining. One animal per group was checked.

- Preparation of slides
To 20 µL of the cell suspension, 180 µL melted low melting point agarose (LMAgarose; Trevigen,
Gaithersburg, USA) was added. The cells were mixed with the LMAgarose and 50 µL was layered on
a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared. The slides
were marked with the study identification number, animal number and group number. The slides were
incubated for 10-15 minutes in the refrigerator in the dark until a clear ring appears at the edge of the
Comet slide area.

- Lysis, electrophoresis and staining of the slides
The cells on the slides were overnight (approximately 17 h) immersed in prechilled lysis solution
(Trevigen) in the refrigerator. After this incubation period, the slides were immersed/rinsed in
neutralization buffer (0.4M Tris-HCl pH 7.4) for approximately 5 minutes. The slides were then placed
in freshly prepared alkaline solution for 30 minutes at room temperature in the dark.
The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the
voltage was set to 1 Volt/cm. The electrophoresis was performed for 30 minutes under constant
cooling (actual temperature 5.5 – 7.0°C). After completion of electrophoresis, the slides were
immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for 5 minutes in
70% ethanol and allowed to dry at room temperature. The slides were stained for approximately 5
minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the
refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room
temperature in the dark.

- Comet scoring
To prevent bias, slides were randomly coded before examination of the Comets. An adhesive label
with study identification number and code were placed over the marked slide. The slides were
examined with a fluorescence microscope connected to a Comet Assay IV image analysis system
(Perceptive instruments Ltd, Suffolk, United Kingdom).

One hundred fifty Comets per slide (50 comets of each replicate LMAgarose circle) were examined.
On a few slides the number of cells was limited, therefore an agarose circle from the second backup
slide was used for scoring.

The following criteria for scoring of Comets were used:
- Only horizontal orientated Comets were scored, with the head on the left and the tail on the right.
- Cells that showed overlap or were not sharp were not scored.
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleated polychromatic erythrocytes
Toxicity:
no effects
Remarks:
The animals of the all groups treated with 500, 1000 and 2000 mg Everzol Orange ED-G Crude/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Sex:
male
Genotoxicity:
negative
Remarks:
Comet slide analysis
Toxicity:
no effects
Remarks:
The animals of the all groups treated with 500, 1000 and 2000 mg Everzol Orange ED-G Crude/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Micronucleated polychromatic erythrocytes
The mean number of micronucleated polychromatic erythrocytes per group and the mean ratio of
polychromatic to normochromatic erythrocytes are presented in Table 2 (APPENDIX 1 ). The individual
data are described in Table 6 (APPENDIX 1 ). The mean number of micronucleated polychromatic
erythrocytes scored in Everzol Orange ED-G Crude treated groups were compared with the
corresponding solvent control group.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the
bone marrow of Everzol Orange ED-G Crude treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control
animals was within the historical solvent control data range (APPENDIX 8).
Cyclophosphamide, the positive control item, induced a statistically significant 46-fold increase in the
number of micronucleated polychromatic erythrocytes (APPENDIX 4). Hence, the acceptability criteria
of the test were met

- Ratio polychromatic to normochromatic erythrocytes
The animals of the groups, which were treated with Everzol Orange ED-G Crude showed no decrease
in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of
this test item on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed
an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating
toxic effects on erythropoiesis.


Comet Slide analysis
After treatment, single cell suspensions from the liver and stomach were prepared. The viability of one
single cell suspension per tissue per group was assessed by using trypan blue. The viability of all
single suspension was 100% (Table 3; APPENDIX 1).

Comet slides were prepared and analysed. An overview of the mean Tail Intensity is presented in
Table 4 - 5 and Figure 1 - 2 (APPENDIX 1). The detailed data of the treatment groups is presented in
APPENDIX 2. The detailed data of the individual rats and slides is presented in APPENDIX 6-7. The
historical data is presented in APPENDIX 8.

- Liver
A statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of Everzol
Orange ED-G Crude-treated male animals at 1000 and 2000 mg/kg compared to the vehicle treated
animals (p<0.05 Dunnet’s t test). The mean Tail Intensity (%) in liver cells of vehicle treated male rats
was 1.89 ± 0.30% (Mean±SD). The Tail Intensities at 500, 1000 and 2000 mg/kg were 2.52 ± 0.42%,
3.59 ± 1.21 and 2.94 ± 0.49 (Mean ± SD), respectively. Since all values were clearly within the
historical vehicle control data range (4.21 ± 3.08%, Mean ± SD) and no dose related response was
shown, the statistically significant increase observed at 1000 and 2000 mg was considered biologically
not relevant and caused by the low variation in the vehicle control data.

The positive control EMS showed a mean Tail Intensity of 93.73 ± 3.14% (Mean ± SD, 49.6-fold
statistically significant induction; Students t test p<0.001). The negative and positive control Tail
Intensities were within the historical control data range. Hence, all criteria for an acceptable assay
were met.

- Stomach
No statistically significant increase in the mean Tail Intensity was observed in the stomach of
Everzol Orange ED-G Crude-treated male animals at any of the dose levels tested compared to the
vehicle treated animals.

The mean Tail Intensity (%) in stomach cells of vehicle treated male animals was 27.81± 10.65%
(Mean ± SD). The positive control EMS, showed a mean Tail Intensity of 97.93 ± 1.10% (Mean ± SD,
3.5-fold statistically significant induction; Students t test p<0.001; APPENDIX 4). The negative and
positive control Tail Intensities were within the historical control data range. Hence, all criteria for an
acceptable assay were met
Conclusions:
It is concluded that the micronucleus test was valid and that Everzol Orange ED-G Crude is not
clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of
2000 mg/kg kg (the maximum recommended dose in accordance with current regulatory guidelines)
under the experimental conditions described in this report. Moreover, Everzol Orange ED-G Crude
does not provoke DNA damage in the Comet assay in liver and stomach cells under the experimental
conditions described in this report.
Executive summary:

The compound Everzol Orange ED-G Crude was tested in the combined alkalinein vivoComet assay
in male rats, to evaluate its potential to induce genotoxic effects.


The study procedures described in this report were based on the most recent OECD and EC
guidelines.

Batch F8411502 of Everzol Orange ED-G Crude was a black powder. The test item was suspended in
Milli-Ro water.

Formulation analysis was performed to determine the accuracy of preparation, homogeneity and
stability of the test substance in formulations. The concentrations analysed in the formulations were
considered in agreement with target concentrations (i.e. mean accuracies between 111% and 114%) .
No test item was detected in the vehicle control samples. The formulations were homogeneous (i.e.
coefficient of variation <1.6%) and stable when stored at room temperature under normal laboratory
light conditions for at least 4 hours.

In the dose range finding study 3 males were dosed once daily via oral gavage with 2000 mg Everzol
Orange ED-G Crude per kg body weight for three consecutive days. The animals showed no treatment
related clinical signs or mortality after dosing.

In the main study, groups of 5 male animals were dosed once daily via oral gavage with vehicle or with
2000, 1000 and 500 mg Everzol Orange ED-G Crude per kg body weight for three consecutive days.
A positive control group (5 male rats) for the comet assays was dosed twice by oral gavage with 200
mg Ethyl Methane Sulfonate (EMS) per kg body weight and a positive control group (5 male rats) for
the micronucleus assay was dosed once by oral gavage with 20 mg cyclophosphamide (CP) per kg
body weight. The animals showed no treatment related clinical signs or mortality after dosing.

Approximately 3-4 hours after the third dose of the vehicle or Everzol Orange ED-G Crude, liver and
stomach tissue were collected. The animals were sacrificed by abdominal aorta bleeding under
isoflurane anaesthesia and liver and stomach tissue was isolated. Single cell suspensions from the
liver and stomach were made followed by Comet slide preparation. The slides were analyzed and the
Tail Intensity (%) was assessed. Bone marrow smears were prepared for micronucleus analysis.

Bone marrow smears were analysed. No increase in the mean frequency of micronucleated
polychromatic erythrocytes was observed in the bone marrow of animals treated with Everzol Orange
ED-G Crude compared to the vehicle treated animals. The incidence of micronucleated polychromatic
erythrocytes in the bone marrow of all negative control animals was within the 95% control limits of the
distribution of the historical negative control database. Cyclophosphamide, the positive control item,
induced a statistically significant 46-fold increase (Studentsttest p<0.05) in the number of
micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic
erythrocytes found in the positive control animals was within the 95% control limits of the distribution of
the historical positive control database. Hence, all criteria for an acceptable assay were met. The
groups that were treated with Everzol Orange ED-G Crude showed no decrease in the ratio of
polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group,
indicating a lack of toxic effects of this test item on erythropoiesis. The group that was treated with
cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic
erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

No biologically relevant increase in the mean Tail Intensity (%) was observed in liver and stomach
cells of Everzol Orange ED-G Crude treated male animals compared to the vehicle treated animals.
The mean Tail Intensity (%) in liver and stomach cells of vehicle treated male animals was 1.89
± 0.30% and 27.81± 10.65% (Mean ± SD), respectively. The positive control EMS showed a mean Tail
Intensity of 93.73 ± 3.14% (Mean ± SD, 49.6-fold statistically significant induction; Studentsttest
p<0.001) and 97.93 ± 1.10% (Mean ± SD, 3.5-fold statistically significant induction; Students t test
p<0.00) in liver and stomach, respectively. The negative and positive control Tail Intensities were
within the historical control data range. Hence, all criteria for an acceptable comet assay were met.

 

It is concluded that the micronucleus test was valid and that Everzol Orange ED-G Crude is not

clastogenic or aneugenic in the bone marrow micronucleus test in male rats up to a dose of

2000 mg/kg kg (the maximum recommended dose in accordance with current regulatory guidelines)

under the experimental conditions described in this report. Moreover, Everzol Orange ED-G Crude

does not provoke DNA damage in the Comet assay in liver and stomach cells under the experimental

conditions described in this report.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The mutagenicity of Everzol Orange ED-G Crude is positive in the bacterial reverse mutation assay, positive in the in vitro mammalian cell chromosomal aberration assay and positive in the in vitro mammalian cell gene mutation assay. 

In two cases, activation with rat and/or hamster S9 mix was necessary to reveal the genotoxic potential. This indicates that this substance is transformed by mammalian enzymes into one or more metabolites that can cause both gene mutations and chromosomal abnormalities.

From the QSAR data, we cannot find similar structure in order to decide the mutagenicity.

As a consequence, we propose in vivo gene mutation tests to verify the mutagenicity.


Justification for selection of genetic toxicity endpoint
Three in vitro mutagenicity tests all showed positive responses. From the QSAR data, we cannot find similar structure in order to judge the mutagenicity. Furthermore, in vivo experiments and human studies are not available. Therefore, it is insufficient to classify mutagenicity.

Justification for classification or non-classification

Based on the available in vivo data, the test substance is not classified for mutagenicity.