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Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 January 2016 - 16 April 2016 (combined micronucleus and Comet assay for liver) and 13 November 2018 - 5 December 2018 (Comet assay, stomach)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In a GLP-compliant combined OECD Guidelines 474 and 489 study, the compound 2-amylanthraquinone was tested in the combined micronucleus and alkaline in vivo Comet assay in male rats, to evaluate its potential to induce genotoxic effects (study reported in 2016). ECHA considered that results for the Comet assay in glandular stomach did not fulfil the quality criteria based on the relatively high Tail Intensity of the negative control and therefore the Comet assay was repeated in 2018 for the glandular stomach (final report available in January 2019). The new study fulfilled the quality criteria and therefore for glandular stomach only the results of this new Comet study are mentioned in this study summary.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
26 September 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
26 September 2014
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
31 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Adopted 29 July 2016 (used for Comet study with stomach tissue)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Appearance: Yellow viscous liquid
- Storage conditions: At room temperature protected from light
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in PEG-400, stable when stored at room temperature under normal laboratory light conditions for at least 4 hours


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 2-amylanthraquinone was dissolved in PEG-400 (Merck, Darmstadt, Germany). The specific gravity of PEG-400 is 1.125 g/mL. 2-amylanthraquinone concentrations were treated with ultra-sonic waves to
completely dissolve the test item.

Test animals

Species:
rat
Strain:
other: Crl:WI (Han)
Details on species / strain selection:
Strain recommended by international guidelines (e.g. OECD and EC)
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 - 10 weeks
- Age at study initiation: 6 weeks (Comet assay with stomach)
- Weight at study initiation: 149.9 ± 5.7 g (139 – 159 g).
- Weight at study initiation: 147 ± 9.3 g (125 – 163 g) (Comet assay with stomach)
- Assigned to test groups randomly: yes
- Fasting period before study: a limited quantity of food was supplied during the night before dosing (approximately 7 g/rat).
- Housing: Group housing of maximum 5 animals per sex in labeled Macrolon cages (type MIV height 180 mm, length 600 mm and width 330 mm) containing sterilised sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom)
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.6 - 21.8°C
- Temperature (°C): 20.4 - 21.8°C (Comet assay with stomach)
- Humidity (%): 29 - 95%
- Humidity (%): 51 - 54% (Comet assay with stomach)
- Air changes (per hr): ca. 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 15-01-2016 To: 16-04-2016
IN-LIFE DATES: From: 13-11-2018 To: 15-11-2018 (Comet assay with stomach)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: polyethylene glycol
- Concentration of test material in vehicle: 300, 90, 30 and 4 mg/mL
- Amount of vehicle: 5 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test compound.
2-amylanthraquinone was dissolved in PEG-400 (Merck, Darmstadt, Germany). The specific gravity of PEG-400 is 1.13 g/mL. 2-amylanthraquinone concentrations were treated with ultra-sonic waves to completely dissolve the test item. 2-amylanthraquinone concentrations were dosed within 3 hours after preparation.
Duration of treatment / exposure:
Three consecutive days
Frequency of treatment:
Once daily
Post exposure period:
Dose-range finding study: 1-4 days
Main study: none (sacrifice ca. one hour post-administration)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle controls
Dose / conc.:
150 mg/kg bw/day
Dose / conc.:
450 mg/kg bw/day
Dose / conc.:
1 500 mg/kg bw/day
No. of animals per sex per dose:
Dose-range finding study: 1/sex/dose and 3/sex/dose
Main study: 5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
ethylmethanesulphonate (Alkaline Comet Assay) and cyclophosphamide (micronucleus test);
- Route of administration: oral (gavage)
- Doses / concentrations: ethylmethanesulphonate: 200 mg/kg bw, cyclophosphamide 20 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow (micronucleus test), liver and stomach (alkaline Comet assay)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on the results of the dose-range finding study. The animals showed no treatment related clinical signs or mortality after dosing with 1000 mg/kg bw. It was concluded that a dose of 1000 mg/kg bw would be too low as highest dose in the main study. Subsequently, 3 males and 3 females were dosed once daily via oral gavage with 1500 mg 2-amylanthraquinone per kg body weight for three consecutive days. The animals showed the following toxic signs after the second and third dosing: lethargy, hunched posture, rough coat, a swollen belly and did not eat all food provided (but no clear decrease in body weight). Based on the results of the dose range finding study a daily dose level of 1500 mg/kg bw was selected as highest dose for the Micronucleus and Comet main test. Since there were no substantial differences in toxicity between sexes only males were used in the main study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 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 and t=45-47 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 by abdominal aorta bleeding under isoflurane anaesthesia at t = 48-49 h.

DETAILS OF SLIDE PREPARATION:
Micronucleus test:
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 4 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.
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.
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).

Comet Assay:
Liver
The isolation method was based on the publication of Hu et al (2002). A portion of 0.56-0.70 gram from the liver was removed and minced thoroughly on aluminium foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking waterbath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+- and Mg2+-free) and kept on ice.

Stomach
This isolation method for glandular stomach is based on the JaCVAM Comet validation study. The stomach was cut open and washed free from food using cold Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free, Life Technologies, Breda, the Netherlands). The forestomach was removed and discarded. The glandular stomach was stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA (Merck, Darmstadt, Germany).
The glandular stomach was then transferred to a petri-dish on ice containing 10 mL mincing buffer incomplete. The surface epithelia of the glandular epithelia was gently scraped 3-4 times with a cell scraper. This layer was discarded since the lifetime of these cells is very short in the body with a maximum of 3 days. Therefore this layer contains a high amount of apoptotic cells which disturb the interpretation in the Comet assay. Moreover, since the lifetime of these cells is very short it is unlikely that these cells play a role in carcinogenesis.
The glandular stomach was then rinsed with mincing buffer incomplete and transferred to a petri-dish containing 10 mL mincing buffer. The stomach was then scraped multiple times with a cell scraper and the cells were collected in the mincing buffer present in the petri-dish. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution, pH 7.5 (DMSO (Merck) was added immediately before use).
The cell suspension was filtered through a 100 μm Cell Strainer (Falcon, Corning life Sciences, Tewksbury, United States) to purify the cell suspension and collected in a tube and stored on ice.

Slides preparation (liver)
To 20 μL of the cell suspension, 280 μL melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added. The cell suspension was mixed with melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) (1xcell suspension + 14xLMAgarose). Fifty to 60 μL was layered on a precoated Comet slide (Trevigen) in duplicate. Three slides per tissue were prepared (in total 6 agarose circles; 3 for scoring and 3 for backup). The slides were marked with the project and
identification number. The slides were incubated for 10-34 minutes (study plan deviation 2) in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.
The cells on the slides were overnight (approximately 17 - 18 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 24 - 28 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 5.0 – 5.5°C). After completion of electrophoresis, the slides were immersed/rinsed in the neutralization buffer. The slides were subsequently immersed for 5 minutes in Absolut ethanol (≥99.6%) 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.

Slides preparation (stomach)
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 μL was layered on a pre-coated Comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 15-17 minutes in the refrigerator in the dark until a clear ring appears at the edge of the Comet slide area.

The cells on the slides were overnight (approximately 18 h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 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 0.7 Volt/cm. The electrophoresis was performed for 20 minutes under constant cooling (actual temperature 4.5°C). After electrophoresis the slides were immersed/rinsed in
neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for 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 and fixed with a coverslip.

METHOD OF ANALYSIS:
Micronucleus test:
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.

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, one of the agorose circles was damaged or the amount of cells present 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.
Evaluation criteria:
Micronucleus test
A test item is considered positive in the micronucleus test if:
a) At least one of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) Any of the results are outside the 95% control limits of the historical control data range.
A test item is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (Dunnett’s test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) All results are within the 95% control limits of the negative historical control data range.

Comet assay
A test compound is considered positive in the Comet assay (in a tissue) if the following criteria are met:
It induces a biologically as well as a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity. In case of other non-dose-dependent significant increases the data interpretation will be on a case by case base.
A test compound is considered as negative in the Comet assay (in a tissue) if the following criteria are met:
None of the tested concentrations show a statistically significant (Dunnett’s test, one-sided, p < 0.05) dose-dependent increase in percentage Tail Intensity.
Statistics:
ToxRat Professional v3.2.1 was used for statistical analysis.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
150 and 450 mg/kg bw: no treatment related clinical signs of toxicity or mortality. 1500 mg/kg bw: a swollen belly, lethargy, diarhea, food avoidance, hunched posture
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: In a dose range finding study 8 animals (group A: 1 male and 1 female animal and group B: 3 male and 3 female animals) were dosed via oral gavage with 1000 and 1500 mg/kg body weight (groups A and B, respectively) of 2-amylanthraquinone once daily for three consecutive days.
- Clinical signs of toxicity in test animals: The animals showed no treatment related clinical signs or mortality after dosing with 1000 mg/kg/day. It was concluded that a dose of 1000 mg/kg would be too low as highest dose in the main study. Subsequently, 3 males and 3 females were dosed once daily via oral gavage with 1500 mg 2-amylanthraquinone per kg body weight for three consecutive days. The animals showed the following toxic signs after the second and third dosing: lethargy, hunched posture, rough coat, a
swollen belly and did not eat all food provided (but no clear decrease in body weight). Based on the results of the dose range finding study a dose level of 1500 mg/kg body weight was selected as highest dose for Micronucleus and Comet main test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of 2-amylanthraquinone 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. Cyclophosphamide, the positive control item, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups, which were treated with 2-amylanthraquinone 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:
Liver:
No statistically significant increase in the mean Tail Intensity (%) was observed in liver cells of 2-amylanthraquinone-treated male animals at any of the dose levels tested compared to the vehicle treated animals. The mean Tail Intensity (%) in liver cells of vehicle treated male rats was 3.68%. The positive control EMS, showed a mean Tail Intensity of 93.25% (25-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 glandular stomach cells of 2-amylanthraquinone treated male treated animals compared to the vehicle treated animals.
The mean Tail Intensity in glandular stomach cells of vehicle-treated rats was 2.24 ± 0.86% (mean ± SD) which is within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a statistically significant increase (Students t test, p<0.001) and showed a mean Tail Intensity of 51.72 ± 6.24% (mean ± SD). The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the MTD. Hence, all criteria for an acceptable assay were met.

Any other information on results incl. tables

Table 1. Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes

Group

Treatment

Dose (mg/kg

Number of micronucleated

Ratio polychromatic/

 

 

body weight)

polychromatic erythrocytes

normochromatic

 

 

 

 

(mean ± S.D.)

(1,2)

erythrocytes

 

 

 

 

 

 

 

(mean ± S.D.)(1,3)

 

 

 

 

 

 

 

 

 

A

Vehicle control

 

0

2.8

±1.1

 

0.97

±0.05

B

2-amylanthraquinone

1500

2.8

±0.8

 

0.91

±0.06

C

2-amylanthraquinone

450

4.0

±1.0

 

1.00

±0.07

D

2-amylanthraquinone

150

3.6

±1.5

 

0.96

±0.06

F

CP

 

20

32.2

±7.8(4)

 

0.30

±0.07

Vehicle control = PEG-400

CP = Cyclophosphamide.

(1)    Five animals per treatment group.

 (2)    At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%

(3)    The ratio was determined from at least the first 1000 erythrocytes counted.

(4)    Significantly different from corresponding control group (Studentsttest, p < 0.001).

Table 2

Overview Tail Intensity in liver cells of male Rats

 

 

 

 

 

 

 

Tail Intensity (%)(1)

S.D.

 

Vehicle Control

 

3.68

1.04

 

2-amylanthraquinone 1500 mg/kg

3.79

1.35

 

2-amylanthraquinone 450 mg/kg

2.89

1.36

 

2-amylanthraquinone 150 mg/kg

2.34

0.84

 

EMS 200 mg/kg

93.25***

2.01

 

(1) Five animals per treatment group.

 

 

 

* = p < 0.001 (assessed with the Student’s t test)

 

 

Vehicle Control = PEG-400

 

 

 

EMS = Ethyl Methanesulfonate

 

 

 

Table 3

Overview Tail Intensity in stomach cells of male Rats

 

 

 

 

 

 

 

Tail Intensity (%)(1)

S.D.

 

Vehicle Control

 

2.24

0.86

 

2-amylanthraquinone 1500 mg/kg

2.47

0.35

 

2-amylanthraquinone 450 mg/kg

2.72

0.75

 

2-amylanthraquinone 150 mg/kg

4.10

1.62

 

EMS 200 mg/kg

51.72***

6.24

 

(1) Five animals per treatment group.

 

 

 

*** = p < 0.001 (assessed with the Student’s t test)

Vehicle Control = PEG-400

EMS = Ethyl Methanesulfonate

Table 4. Historical control data micronucleus assay (negative control)

 

Number of micronucleated polychromatic erythrocytes per

 

2000 polychromatic erythrocytes

 

males

 

 

Range

0 - 5

 

 

Mean

1.8

 

 

SD

1.1

 

 

n

100

SD = Standard deviation

n = Number of observations

 Historical control data from experiments performed between November 2004 and November 2014.

Table 5. Historical data micronucleus assay (cyclophosphamide)

 

Number of micronucleated polychromatic erythrocytes per

 

2000 polychromatic erythrocytes

 

males

 

 

Range

14 - 61

 

 

Mean

33.2

 

 

SD

12.7

 

 

n

98

 

Number of micronucleated polychromatic erythrocytes per

 

2000 polychromatic erythrocytes

 

males

 

 

Range

14 - 61

 

 

Mean

33.2

 

 

SD

12.7

 

 

n

98

 

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between November 2004 and November 2014.

Table 6. Historical data Comet assay (physicological saline orally dosed for three consecutive days)

 

Liver Tail Intensity (%)

Stomach Tail Intensity (%)

 

Males and Females

Males and Females

 Range 0.13 - 21.13   

Lower control limit

(95% control limits)

   -2.26

Upper control limit

(95% control limits)

  12.32 
 Mean  4.21  5.10
 SD  3.08  7.54
 n  65  65

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 - 2015 (liver)

Historical control data from experiments performed in January - November 2018 (stomach)

Table 7. Historical data Comet assay (200 mg/kg EMS orally dosed for two consecutive days)

 

Liver Tail Intensity (%)

Stomach Tail Intensity (%)

 

Males and Females

Males and Females

Range

31.32 – 98.09

  Lower control limit

(95% control limits)

 

40.25 

Upper control limit

(95% control limits)

77.72

 

Mean

78.40 

 58.89

SD

18.32

14.06

 

 

 

n

60

64

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed in 2012 - 2015 (liver)

Historical control data from experiments performed in January - November 2018 (stomach)

Applicant's summary and conclusion

Conclusions:
The test substance 2-amylanthraquinone did not induce an increase in the mean frequency of micronucleated polychromatic erythrocytes in the bone marrow or an increase in the mean tail intensity in liver and stomach cells in a combined micronucleus/alkaline Comet assay in rats administered the test substance by oral gavage for three consecutive days up to the maximal dose level of 1500 mg/kg bw/day.
Executive summary:

In a GLP-compliant combined OECD Guidelines 474 and 489 study, the compound 2-amylanthraquinone was tested in the combined micronucleus and alkaline in vivo Comet assay in male rats, to evaluate its potential to induce genotoxic effects (study reported in 2016). ECHA considered that results for the Comet assay in glandular stomach did not fulfil the quality criteria based on the relatively high Tail Intensity of the negative control and therefore the Comet assay was repeated in 2018 for the glandular stomach (final report available in January 2019). The new study fulfilled the quality criteria.

Based on the dose-range finding study, the highest dose level was chosen to be 1500 mg/kg bw/day. In the main study, 5 males/dose were dosed by oral gavage with vehicle or with 1500, 450 and 150 mg/kg bw/day for three consecutive days. The animals of the groups treated with 150 and 450 mg/kg bw/day and the animals of the negative and positive control groups showed no treatment related signs of toxicity or mortality. The following treatment related sign of toxicity were made in the groups treated with 1500 mg/kg bw/day: swollen belly, lethargy, diarhea and hunched posture. Approximately 3-4 hours after the third dose of the vehicle or 2-amylanthraquinone, liver and stomach tissue were collected for the Comet assay and femurs were collected for the micronucleus test. No statistically significant and biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with 2-amylanthraquinone. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. No statistically significant increase in the mean Tail Intensity (%) was observed in liver and stomach cells of 2-amylanthraquinone treated rats at any of the dose levels tested compared to the vehicle treated animals. The positive controls ethylmethane sulphonate and cyclophosphamide produced satisfactory results. It is concluded that the micronucleus test was valid and that 2-amylanthraquinone is not clastogenic or aneugenic in the bone marrow micronucleus test in rats up to and including a dose of 1500 mg/kg bw/day (the maximum tolerated dose) under the experimental conditions described in the report. Moreover, 2-amylanthraquinone does not provoke DNA damage in the Comet assay in liver and stomach cells up to and including a dose of 1500 mg/kg bw/day under the experimental conditions described in the reports.