Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 (OECD TG471 and EU Method B.13/14).

 

Everzol Red CDN Crude is mutagenicunder the condition of in vitro mammalian cell gene mutation test(OECD TG476).

Link to relevant study records

Referenceopen allclose all

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
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 or hamster liver S9-mix
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
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
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
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
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
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
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

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

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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 17, 2015 to September 03, 2015
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:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9 activation system
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
methylmethanesulfonate
Evaluation criteria:
A two-fold increase in mutant frequency over negative control/or a concentration related response would be considered a positive response. The result did not meet the criteria above would be judged as negative.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid

Table 1. Relative Total Growth (RTG) of Everzol Red CDN Crude 4-hour Treated Cells in the Absence of S9 Activation

Treatment

(μg/mL)

Replicate culture

SGc

RSG (%)d

RPE (%)e

RTG (%)f

Aversge RTG (%)g

N.C.a

(1)

41.73

100.00

100.00

100.00

100.00

N.C.a

(2)

41.99

100.00

100.00

100.00

40

(1)

40.95

97.83

85.17

83.32

80.62

40

(2)

42.04

100.43

77.59

77.92

79

(1)

42.66

101.91

94.42

96.22

97.51

79

(2)

42.87

102.41

96.47

98.79

157

(1)

39.18

93.60

84.00

78.62

82.86

157

(2)

39.51

94.39

92.28

87.10

313

(1)

36.70

87.67

100.49

88.10

80.62

313

(2)

36.69

87.65

83.44

73.14

625

(1)

30.55

72.98

94.38

68.88

67.32

625

(2)

29.36

70.14

93.75

65.76

1250

(1)

8.33

19.90

47.39

9.43

8.95

1250

(2)

7.81

18.66

45.36

8.46

P.C.b

(1)

41.73

99.69

64.09

63.89

62.75

P.C.b

(2)

38.78

92.64

66.51

61.61

a: Negative control: culture medium

b: Positive control in non-activation system: 10 μg/mL Methyl Methanesulfonate (MMS)

c: SG: Suspension Growth = [cell density of 24 hours after treatment (before adjustment) / cell density of 0 hour after treatment (after adjustment)] × [cell density of 48 hours after treatment (before adjustment) / cell density of 24 hours after treatment (after adjustment)]

d: RSG (%): Relative Suspension Growth = SG(negative control)/ SG(negative control)× 100

Relative Suspension Growth = SG(test)/ SG(average of negative control)× 100

e: RPE (%) : Relative Plating Efficiency = Plating Efficiency(negative control)/Plating Efficiency(negative control)×100

Relative Plating Efficiency = Plating Effciency(test)/Plating Efficiency(average of negative control)×100

f: RTG (%): Relative Total Growth = [RSG (%) × RPE (%)]/100

g: Average RTG (Relative Total Growth) = [RTG(treatment (1))+ RTG(treatment (2))] /2

Table 2. Relative Total Growth (RTG) of Everzol Red CDN Crude 4-hour Treated Cells in the Presence of S9 Activation

Treatment

(μg/mL)

Replicate culture

SGc

RSG (%)d

RPE (%)e

RTG (%)f

Aversge RTG (%)g

N.C.a

(1)

37.70

100.00

100.00

100.00

100.00

N.C.a

(2)

38.67

100.00

100.00

100.00

79

(1)

35.51

92.99

98.06

91.19

101.82

79

(2)

35.42

92.76

121.23

112.45

157

(1)

34.05

89.17

118.08

105.29

105.60

157

(2)

35.94

94.12

112.53

105.91

313

(1)

31.13

81.52

120.13

97.93

92.42

313

(2)

35.51

92.99

93.46

86.91

625

(1)

28.09

73.56

113.31

83.35

76.75

625

(2)

26.05

68.22

102.82

70.14

1250

(1)

8.76

22.94

75.61

17.34

15.18

1250

(2)

8.41

22.02

59.15

13.02

2500

(1)

3.70

9.69

4.04

0.39

0.29

2500

(2)

3.84

10.06

1.86

0.19

P.C.b

(1)

25.11

65.76

97.90

64.38

56.04

P.C.b

(2)

23.48

61.49

77.58

47.70

a: Negative control: culture medium

b: Positive control in non-activation system:200 μg/mL 2-acetylaminofluorine (2AAF)

c: SG: Suspension Growth = [cell density of 24 hours after treatment (before adjustment) / cell density of 0 hour after treatment (after adjustment)] × [cell density of 48 hours after treatment (before adjustment) / cell density of 24 hours after treatment (after adjustment)]

d: RSG (%): Relative Suspension Growth = SG(negative control)/ SG(negative control)× 100

Relative Suspension Growth = SG(test)/ SG(average of negative control)× 100

e: RPE (%) : Relative Plating Efficiency = Plating Efficiency(negative control)/Plating Efficiency(negative control)×100

Relative Plating Efficiency = Plating Efficiency(test)/Plating Efficiency(average of negative control)×100

f: RTG (%): Relative Total Growth = [RSG (%) × RPE (%)]/100

g: Average RTG (Relative Total Growth) = [RTG(treatment (1))+ RTG(treatment (2))] /2

 

Table 3. Summary of Average Mutant Frequency of Everzol Red CDN Crude Treated Cells after 12-13 Days Selection Period

Treatment

(μg/mL)

Average RTG (%)

P.E. %d

M.F. (×10-6)eof Total Colonies

M.F. (×10-6)eof Large Colonies

M.F. (×10-6)eof Small Colonies

Without S9 activation system (4-hours)

N.C.a

100.0

93.60 ± 13.62

68.68 ± 19.03

3.03 ± 3.32

64.22 ± 18.60

40

80.62

76.17 ± 9.62

84.73 ± 17.32

8.34 ± 0.92

75.58 ± 16.49

79

97.51

89.34 ± 9.33

67.22 ± 13.07

8.02 ± 6.82

58.48 ± 11.35

157

82.86

82.49 ± 10.01

82.37 ± 45.82

6.76 ± 6.16

74.71 ± 39.49

313

80.62

86.08 ± 10.27

75.07 ± 24.02

8.74 ± 3.78

64.01 ± 21.44

625

67.32

88.04 ± 9.29

98.43 ± 31.06

8.46 ± 3.29

83.19 ± 27.30

1250

8.95

43.41 ± 6.61

197.17* ± 53.53

15.28 ± 15.08

177.48 ± 51.89

P.C.b

62.75

61.12 ± 3.82

533.97** ± 56.92

24.09 ± 7.85

487.19 ± 67.82

With S9 activation system (4-hours)

N.C.a

100.00

79.70 ± 13.48

95.42 ± 24.78

7.02 ± 7.15

86.32 ± 26.61

79

101.82

87.38 ± 10.56

107.07 ± 55.59

8.59 ± 5.55

95.82 ± 50.62

157

105.60

91.90 ± 8.62

83.76 ± 25.46

8.16 ± 5.85

73.43 ± 22.52

313

92.42

85.11 ± 16.34

101.35 ± 29.59

7.30 ± 5.00

91.65 ± 24.87

625

76.75

86.13 ± 6.91

126.88 ± 31.90

18.56 ± 9.26

105.25 ± 25.84

1250

15.18

53.70 ± 12.03

229.50* ± 81.82

19.80 ± 12.59

205.81 ± 67.31

2500

0.29

2.35 ± 1.35

697.92* ± 1642.72

0.00 ± 0.00

697.92 ± 1642.72

P.C.c

56.04

69.93 ± 12.09

594.12** ± 208.06

24.90 ± 6.85

547.97 ± 194.73

a: Negative control: culture medium

b: Positive control in non-activation system: 10 μg/mL Methyl Methanesulfonate (MMS)

c: Positive control in S9 activation system: 200 μg/mL 2-acetylaminofluorine (2AAF)

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

e: Mutant Frequency (M.F.) = C.E.% in selective medium / P.E.% in non-selective medium

*: M.F. of total colonies for treatment group was two-fold over than M.F. of total colonies for negative control.

**: M.F. of total colonies for positive control was five-fold over than M.F. of total colonies for negative control.

Conclusions:
According to OECD 476 test method, Everzol Red CDN Crude is mutagenic in the In Vitro Mammalian Cell Gene Mutation Test.
Executive summary:

This test using the procedures outlined in the QPS Taiwan Study Plan for T65314001-GT and OECD 476 (OECD, 1997).The results of this OECD 476 test forEverzol Red CDN Crudeshow that test validity criteria was met.

Based on the results of the solubility and toxicity results in the preliminary assay, the highest dose were 1250 and 2500 μg/mL respectively in the non-activation system and the S9-activation system.In the gene mutation test, six doses of Everzol Red CDN Crude, negative and positive controls were tested in repetitions with or without S9 activation system. For mutagenicity evaluation, in the non-activation system, the average M.F. at 1250 μg/mL was more than two-fold of negative control. In the S9 activation system, the average M.F. at 1250 and 2500 μg/mL were also more than two-fold of negative control. With the acceptable range of cytotoxicity, 1250 μg/mL in the S9 activation system was the concentration for evaluating the mutagenicity and there was a dose-response relationship. The M.F. of L5178Y/TK+/-cells treated with Everzol Red CDN Crude showed positive in the presence of metabolic system. Therefore, under these test conditions, the test article Everzol Red CDN 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 Red CDN Crude did not show any evidence of causing an increase in DNA strand breaks in the liver of male Crl:CD(SD) rats administered Everzol Red CDN Crudeorally by gavage in this in vivo test procedure.

Everzol Red CDN Crude has shown evidence of causing an increase in DNA strand breaks in the duodenum and glandular stomach of male Crl:CD(SD) rats when administered orally by gavage in this in vivo test procedure. Seen from the results of other evidence, test substance is unlikely that the DNA strand breaks observed in the duodenum and glandular stomach are representative of a true genotoxic response.

Everzol Red CDN Crudehas not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male Crl.CD(SD) rats when administered orally by gavage in this in vivo test procedure.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 28, 2019 to June 24, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male
Details on test animals and environmental conditions:
Preliminary toxicity test: Males weighed between 183 g - 190 g.
Females weighed between 176 g - 177 g.
Micronucleus test: Males weighed between 167 g - 206 g.
Animal age on dispatch and on Day 1 of dosing was:
Preliminary toxicity test: On Dispatch Males and females ca 42-48 days old.
Day 1 Males and females ca 47-53 days old.
Micronucleus test: On Dispatch Males ca 42-48 days old.
Day 1 Males ca 47-53 days old.

After arrival the weight of the animals was checked and found to be acceptable. The animals were randomly assigned to groups and given a unique tail mark. Each group was kept with the sexes separated in cages. The animals were kept in a controlled environment with the thermostat and relative humidity within target ranges of 20 to 24°C and 40 to 70% respectively, throughout the study. The room was illuminated by artificial light for 12 hours per day.
All animals were allowed free access to pelleted Envigo Teklad 2014C diet and tap water ad libitum.
Route of administration:
oral: gavage
Vehicle:
purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Preliminary Toxicity Test concentration: 200 mg/ml
Main test concentration: 50, 100, 200 mg/ml
Duration of treatment / exposure:
The test item was administered on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, 3 hours before sampling.
Dose / conc.:
500 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
2 000 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)
The positive control for the Comet test was Ethyl Methanesulphonate (EMS; CAS no. 62-50-0) 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:
Following electrophoresis three slides per animal per tissue were analysed for comets. Slides were visualised by staining with SYBR GOLD® via fluorescence microscopy. 150 morphologically normal cells were analysed for the presence of comets per animal per tissue.
All tissue samples were prepared using identical methods, the cause of the hedgehog cells is not considered to be mechanical/enzyme induced damaged during sample preparation. In addition to this, the potent genotoxin EMS (positive control for the comet phase) caused large increase in the % TI in all tissues analyzed.

Comet Phase
Glass slides were dipped in 1% normal melting point agarose and left to air dry prior to addition of the cell suspension layer.
Sections of the liver, glandular stomach and duodenum were placed into ice cold mincing solution, all samples were stored on ice before processing for Comet analysis. Single cell suspensions were prepared using a tissue specific method.

For each tissue type, an appropriate dilution of the cell suspensions were made and mixed with the appropriate volume of 0.5% low melting point agarose. A 75µL aliquot of the cell/agar mix was dispensed onto the appropriate pre-dipped slides and cover-slipped.
Comet slides were prepared from all cell suspensions.
Once the agar had set the cover slips were removed and the slides immersed in chilled lysis solution in a light proof box. These were stored at 2 - 8ºC overnight prior to electrophoresis.

Micronucleus Phase
One femur was dissected out from each animal. The femurs were cleaned of all excess tissue and blood and the heads of the femurs removed from each bone. The bone marrow of one femur from each animal was flushed out and pooled in a total volume of 3 mL of filtered foetal bovine calf serum by aspiration.
The resulting cell suspensions were centrifuged at 1000 rpm (150 g) for 5 minutes and the supernatant discarded. The final cell pellet was resuspended in a small volume of foetal bovine calf serum to facilitate smearing in the conventional manner on glass microscope slides (Schmid 1976).
The slides were fixed in methanol and allowed to air dry. They were then rinsed in purified water and stained using an acridine orange solution at 0.0125 mg/mL and stored at room temperature in the dark until required. Prior to scoring the slides were wet mounted with coverslips using purified water.

Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Micronucleated polychromatic erythrocytes
Toxicity:
no effects
Remarks:
Animals were treated with Everzol Red CDN Crude at dose levels of 500, 1000 and 2000 mg/kg/day. No clinical signs of toxicity were observed for Everzol Red CDN Crude at any dose level.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Sex:
male
Genotoxicity:
negative
Remarks:
Comet slide analysis
Toxicity:
yes
Remarks:
Cytotoxic changes related to treatment with Everzol Red CDN Crude were observed in the stomach (inflammation) and duodenum (degeneration) at 1000 and 2000 mg/kg/day.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose levels of 500, 1000 and 2000 mg/kg/day were selected for the main test.
The main test was carried out in male animals only. No mortalities were observed throughout the duration of the test. Detailed body weights are presented in Appendix 1.
Table 1 gives a summary of the results and statistical analysis of the comet phase. The results for individual animals for the comet phase are presented in Table 2 and hedgehog cell data are presented in Table 3.
Table 4 gives a summary of the results and statistical analysis of the micronucleus phase. The results for individual animals for the micronucleus phase are presented in Table 5.

----Cinical Signs------------
No clinical signs of toxicity were observed for the vehicle control, and positive control or animals administered Everzol Red CDN Crude at any dose level.
Small incidences of body weight loss were observed throughout the main test.

----Tail Intensity------------
The vehicle control group mean % TI values for the liver, duodenum and glandular stomach of Crl:CD(SD) rats were within the 95% confidence limits of the current vehicle historical control range for the individual tissues.
The positive control compound, EMS, produced a statistically significant increase (p<0.001) in the group median % TI when compared to the vehicle control values in all tissues analysed and all % TI values were comparable to the historical positive control range.
There were no statistically significant increases in the group median % TI in the liver of male Crl:CD(SD) rats administered Everzol Red CDN crude at 500, 1000 or 2000 mg/kg/day, compared to vehicle control values. The group mean and median % TI values from all treatment groups were within the 95% confidence limits of the current vehicle historical control range.
There were no statistically significant increases in the group median % TI in the duodenum and glandular stomach of male Crl:CD(SD) rats administered Everzol Red CDN crude at 500 mg/kg/day, compared to vehicle control values. The group mean, and median % TI values were within the 95% confidence limits of the current vehicle historical control range.
There was a statistically significant increase (p<0.001) in the group median % TI observed in the duodenum and glandular stomach of male Crl.CD(SD) rats administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day. The group mean % TI values for animals administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day were within the 95% confidence limits of the current vehicle historical control range.

----Hedgehog Cell------------
There were no hedgehog cells observed in the liver of male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level.
There was a significant increase in the number of hedgehog cells observed in the duodenum and glandular stomach of male Crl:CD(SD) rats administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day, compared to the concurrent vehicle control

----Micronucleated Polychromatic Erythrocyte Counts (MPCE)------
There were no statistically significant increases in the group %MPCE observed in male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level, compared to vehicle control values. All group mean values were within the current vehicle historical range (95% confidence limits).

----Micronucleated Normochromatic Erythrocyte (MNCE)------
There were no substantial increases in the incidence of micronucleated normochromatic erythrocytes in male Crl:CD(SD) rats administered Everzol Red CDN Crude at any dose level.

----Proportion of Polychromatic Erythrocytes (%PCE)-------
There were no statistically significant decreases in the group %PCE observed in male Crl:CD(SD) rats administered Everzol Red CDN crude at any dose level, compared to vehicle control values. The group mean %PCE values were all within the current vehicle historical control range (95% confidence limits).

----Histopathology-------
cytotoxic changes related to treatment with Everzol Red CDN Crude were observed in the stomach (inflammation) and duodenum (degeneration) at 1000 and 2000 mg/kg/day.

Conclusions:
Comet Phase

It is concluded that Everzol Red CDN Crude did not show any evidence of causing an increase in DNA strand breaks in the liver of male Crl:CD(SD) rats administered Everzol Red CDN Crude orally by gavage in this in vivo test procedure.
It is concluded that Everzol Red CDN Crude has shown evidence of causing an increase in DNA strand breaks in the duodenum and glandular stomach of male Crl:CD(SD) rats when administered orally by gavage in this in vivo test procedure. However, these increases in DNA strand breaks are directly associated with an increase in hedgehog cells; although the exact cause of these cellular phenomena is uncertain, it is clear that this would have an impact on the comet response. In addition, histopathology showed evidence of cytotoxic changes related to treatment with Everzol Red CDN Crude which were consistent with the increase in DNA damage seen in the comet assay. Therefore, it is unlikely that the DNA strand breaks observed in the duodenum and glandular stomach are representative of a true genotoxic response.
Micronucleus Phase

It is concluded that Everzol Red CDN Crude has not shown any evidence of causing an increase in the induction of micronucleated polychromatic erythrocytes or bone marrow cell toxicity in male Crl.CD(SD) rats when administered orally by gavage in this in vivo test procedure.
Executive summary:

This study was designed to assess the potential of Everzol Red CDN Crude to induce DNA strand breaks in the liver, glandular stomach and duodenum of Crl: CD(SD) rats and also assess the potential induction of micronuclei by Everzol Red CDN Crude in the bone marrow cells of Crl:CD(SD) rats.

Animals were treated with Everzol Red CDN Crude orally by gavage on three occasions, the second dose being administered approximately 24 hours after the first dose, with the third dose being administered approximately 21 hours after the second dose, 3 hours before sampling.

The vehicle control group received water (purified by reverse osmosis) and the positive control group for the comet phase receivedEthyl methanesulphonate(EMS)at 200 mg/kg,dosed on a single occasion,approximately 3 hours before sampling.

All animals in the vehicle control, test item dose groups and the positive controls were dosed orally by gavage using a dose volume of 10 mL/kg.

On the basis of results from the preliminary toxicity test, dose levels of 500, 1000 and 2000 mg/kg/day were selected for the main test. No substantial differences in toxicity were observed between the sexes in the preliminary toxicity test, therefore, in line with current guidelines the main test was performed using male animals only.

Comet Phase

Cell suspensions from the liver, glandular stomach and duodenum were obtained fromanimals in the vehicle control group and in each of the test item groups approximately 3 hours after administration of the third dose. Cell suspensions from animals in the positive control group were obtained approximately 3 hours after a single dose.

DNA strand breaks were assessed by comparing the group mean and median % tail intensities (% TI) from Everzol Red CDN Crude treated animals with the concurrent vehicle control values. The slides were also examined for any overt toxicity, e.g. an increase in background debris and/or an increase in the incidence of excessively damaged cells
(i.e. Hedgehog cells).
 These cells were excluded from the analysis, along with any cells that had unusual staining artefacts.

Micronucleus Phase

Bone marrow smears were obtained fromanimals in the vehicle control and in each of the test item groups 3 hours after administration of the third dose.

One smear from each animal was examined for the presence of micronuclei in 4000 polychromatic erythrocytes. The proportion of polychromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal. A record of the incidence of micronucleated normochromatic erythrocytes was also kept.

Discussion

 

Hedgehogs cells are heavily damaged cells which exhibit a specific morphology (a small or non-existent head and a large diffuse tail); they are considered to be associated with cytotoxicity, necrosis or apoptosis; however, the exact etiology of hedgehog cells is ambiguous. Other evidence suggests they may be caused by mechanical/enzyme induced damage during sample preparation or extreme effects of test chemical genotoxicity (OECD 489, 2016).

 

There were no hedgehog cells observed in the vehicle control animals; asall tissue samples were prepared using identical methods, the cause of the hedgehog cells is not considered to be mechanical/enzyme induced damaged during sample preparation. In addition to this, the potent genotoxin EMS (positive control for the comet phase) caused large increase in the % TI in all tissues analyzed;however, there were no hedgehog cells observed. Therefore, it is unlikely that the increases in hedgehog cells are due to an extreme effect of test item genotoxicity.

 

The hedgehog cells observed in the duodenum and glandular stomach of male animals administered Everzol Red CDN crude at 1000 and 2000 mg/kg/day are considered to be directly related to the increases in the % TI, further confirmed by the plateau in %TI and hedgehog cells in the glandular stomach. Although the exact cause of these cellular phenomena is uncertain, it is clear that they, or their cause, have in this study compromised the comet response.

 

The guidance is clear that in the presence of hedgehog cells or clear cytotoxicity any relevant increases should be interpreted with care. It is considered that the increases obtained in the duodenum and glandular stomach, are unlikely to be of genotoxic origin.

 

Histopathology performed on dose levels 1000 and 2000 mg/kg/day along with vehicle control samples for the glandular stomach and duodenum confirmed inflammation (stomach) and degeneration (duodenum) were consistent with an increase in DNA damage seen in the comet assay.

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

Additional information

Gene mutation study in bacteria (OECD TG471and EU Method B.13/14)

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 5000 μg/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.

 

Mammalian cell gene mutation tests(OECD TG476)

Based on the results of the solubility and toxicity results in the preliminary assay, the highest dose were 1250 and 2500 μg/mL respectively in the non-activation system and the S9-activation system.In the gene mutation test, six doses of Everzol Red CDN Crude, negative and positive controls were tested inrepetitionswith or without S9 activation system. For mutagenicity evaluation, in the non-activation system, the average M.F. at 1250 μg/mL was more than two-fold of negative control. In the S9 activation system, the average M.F. at 1250 and 2500 μg/mL were also more than two-fold of negative control. With the acceptable range of cytotoxicity, 1250 μg/mL in the S9 activation system was the concentration for evaluating the mutagenicity and there was a dose-response relationship. The M.F. of L5178Y/TK+/-cells treated with Everzol Red CDN Crude showed positive in the presence of metabolic system. Therefore, under these test conditions, the test article Everzol Red CDN Crude is mutagenic in the In Vitro Mammalian Cell Gene Mutation Test.

Everzol Red CDN Crude: Crl:CD(SD) RatIn Vivo Comet Test Including Micronucleus Analysis (OECD TG474/489)

The guidance is clear that in the presence of hedgehog cells or clear cytotoxicity any relevant increases should be interpreted with care. It is considered that the increases obtained in the duodenum and glandular stomach, are unlikely to be of genotoxic origin.

 

Histopathology performed on dose levels 1000 and 2000 mg/kg/day along with vehicle control samples for the glandular stomach and duodenum confirmed inflammation (stomach) and degeneration (duodenum) were consistent with an increase in DNA damage seen in the comet assay.

Justification for classification or non-classification

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