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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 12 October 2004 to 02 December 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report date:
2005

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: In vivo micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[(2-nitrophenyl)amino]ethanol
EC Number:
225-555-8
EC Name:
2-[(2-nitrophenyl)amino]ethanol
Cas Number:
4926-55-0
Molecular formula:
C8H10N2O3
IUPAC Name:
2-[(2-nitrophenyl)amino]ethanol
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, lot no. 17
- Expiration date of the lot/batch: 11 December 2005
- Purity test date:11 December 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light and moisture
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle:The test article was
soluble in the vehicle at all concentrations, including 200 mg/mL, the maximum concentration
tested. Concentrations of 0.4009 and 205.6 mg/mL were stable for 24 hours at room temperature and for 15 days when kept at 5 ± 3°C.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test article dosing formulations were prepared in PEG 400. For each phase of the study, the dose formulations were prepared fresh just prior to use. The appropriate amount of the test article for each concentration was weighed and mixed with the appropriate amount of the vehicle. Each concentration was prepared independently and all appeared to be bright yellow solutions, after vortexing.

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: from Harlan Sprague Dawley, Inc.
- Age at study initiation: 7 to 9 weeks old
- Weight at study initiation: Dose range finding studies : 28.4-32.5 g (males) and 22.0-27.2 g (females) ; definitive micronucleus study : 26.5 to 35.4 g (males), only males were used
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Mice were housed up to five per each rodent Micro-Barrier cage
- Diet (e.g. ad libitum): free access to certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet)
- Water (e.g. ad libitum):free access to tap water (Washington Suburban Sanitary Commission, Potomac Plant)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 deg Celsius
- Humidity (%): 50 ± 20% relative humidity
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air every hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 5 October 2004 (range finding studies) ; 9 November ( Main study) To: 2 December 2004(Main study)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: polyethylene glycol 400 (PEG 400)
- Justification for choice of solvent/vehicle: not specified
- Concentration of test material in vehicle: 25, 50, 100, 200 mg/mL
- Amount of vehicle (if gavage or dermal): 10 mL/kg
- Type and concentration of dispersant aid (if powder): vortexed
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test article dosing formulations were prepared in PEG 400. For each phase of the study, the dose formulations were prepared fresh just prior to use. The appropriate amount of the test article for each concentration was weighed and mixed with the appropriate amount of the vehicle. Each concentration was prepared independently and all appeared to be bright yellow solutions, after vortexing.
Duration of treatment / exposure:
24 hours, 48 hours for high dose group and control
Frequency of treatment:
One single administration
Post exposure period:
24 hours, 48 hours for high dose group and control
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
first dose range finding study
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
first dose range finding study
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
first dose range finding study
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Remarks:
first dose range finding study
Dose / conc.:
1 200 mg/kg bw/day (nominal)
Remarks:
second dose range finding study
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Remarks:
second dose range finding study
Dose / conc.:
1 800 mg/kg bw/day (nominal)
Remarks:
second dose range finding study
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Definitive Micronucleus Study
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Definitive Micronucleus Study
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Definitive Micronucleus Study
No. of animals per sex per dose:
3 animals per dose per sex in dose range finding studies , 5 males for Definitive Micronucleus Study per dose , 3 males per time poit for bleeding (1, 2, 4, 6 and 8 hours post dose)
Control animals:
yes
yes, concurrent vehicle
Positive control(s):
none; no data; cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: oral gavage
- Doses / concentrations: 5mg/ml in distilled water, dose : 50 mg/kg

Examinations

Tissues and cell types examined:
Bonne marrow, erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based on the findings in the pilot study and the toxicity study, doses of 250, 500 and 1000 mg/kg were selected for the definitive study. The high dose was considered to be the maximum tolerated dose producing signs of toxicity such that higher dose levels would be expected to produce lethality. The mid dose was 1/2 the high dose and the low dose was 1/2 the mid dose. Since no differences in the toxicity between male and female mice were observed, only male mice were used in the definitive study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):Bone marrow cells (polychromatic erythrocytes, PCEs), collected 24 and 48 hours after treatment were examined microscopically for presence of micronuclei (MPCEs)

DETAILS OF SLIDE PREPARATION:At the scheduled sacrifice times, 24 and 48 hours postdose, five mice per treatment were sacrificed by CO2 asphyxiation and incision of the diaphragm. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol and stained with May-Gruenwald-Giemsa stain and permanently mounted.

METHOD OF ANALYSIS: Bone marrow cells, polychromatic erythrocytes (PCEs) were analyzed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that are bluish in color and micronuclei are round, dark blue nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte.Using a light microscope and medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes (PCEs/ECs ratio).

OTHER:
Evaluation criteria:
The mean incidence of micronucleated polychromatic erythrocytes must not exceed 10/2000 polychromatic erythrocytes (0.5%) in the negative (vehicle) control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the negative control group (p≤ 0.05, Dunnett’s t-test).
Statistics:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using one way analysis of variance and Dunnett’s t-test (post hoc) for group mean comparison and regression analysis for detecting any trend using square-root transformed data. Any significantly increased response was indicated with the corresponding “p” value where a “p” value ≤ 0.05 was considered significant. All analyses were performed separately for each sampling time. If there were any concentrations with a significant increase, a trend test would have been performed for each group compared to vehicle control group. In order to quantify the test articles effect on erythropoiesis, as a measure of toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group (PCEs/total erythrocyte ratio). All conclusions were based on sound scientific judgment taking into account biological relevance. However, as a guide to interpretation of the data, the test article was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p≤ 0.05, Dunnett’s ttest) at any sampling time.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Pilot Toxicity Study (First Dose Range Finding Study)
In the pilot toxicity study animals (3/sex/dose) were treated with test item at 250, 500, 1000 or 2000 mg/kg. One male and 3 female mice were found dead at 2000 mg/kg. Lethargy and prostration were seen in one male and all females at 2000 mg/kg and convulsions, irregular breathing, ataxia and lacrimation were seen in one female at 2000 mg/kg. All males and all females at 2000 mg/kg had an orange skin tone, and orange urine was noticed in all animals at all doses. Reductions in mean (group) body weights, up to 13%, were observed in some of the test article treated groups during the course of the study.

Toxicity Study (Second Dose Range Finding Study)
Since there was excessive treatment-related toxicity in the pilot study, a toxicity study was
conducted in order to determine the maximum tolerated dose. In the toxicity study, animals
(3/sex/dose) were treated with the test item at 1200, 1500 or 1800 mg/kg. One female mouse at 1200 mg/kg, one male mouse and one female mouse at 1500 mg/kg and all males and females at 1800 mg/kg were found dead within 2 days of observation period. Lethargy, piloerection, and an orange skin tone were observed in one female at 1200 mg/kg, and in all males and females at 1500 and 1800 mg/kg. Orange colored urine was observed in animal cages at all doses. In addition, at 1800 mg/kg, hunched position was observed in all males and females, tremors were observed in one male and two females and ataxia was observed in one male. Slight reductions in mean (group) body weights, up to approximately 2.6 %, were observed in some of the test article treated groups.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): A statistically significant increase in the number of micronucleated PCEs/10000 PCEs scored was not observed in any test article-treated group relative to the vehicle control
- Ratio of PCE/NCE (for Micronucleus assay): reductions, up to 18% (500 mg/kg treatment group) in the ratio of PCEs to total erythrocytes were observed in the test article-treated groups relative to the vehicle control
- Appropriateness of dose levels and route: yes
- Statistical evaluation: yes

Any other information on results incl. tables

Table 1: Pilot Toxicity Study - Body Weight and Mortality Data Following Dose Administration of Test Item ICR Mice

Group Mean BodyWeights(g)                %Change¹

Treatment (10 mL/kg)

Sex

Pretreat

Day 1

Day 2

 

Day 1

Day 2

Mortality²

Test item

250 mg/kg

 

M

 

33.0

 

32.2

 

32.2

 

 

-2.4%

 

-2.4%

 

0/3

 

 

±1.4

±1.2

±1.5

 

 

 

 

 

F

22.8

22.1

21.3

 

-3.1%

-6.6%

0/3

 

 

±1.3

±0.9

±1.4

 

 

 

 

500 mg/kg

M

32.7

33.3

31.8

 

1.8%

-2.8%

0/3

 

 

±1.1

±1.5

±1.6

 

 

 

 

 

F

23.1

22.5

21.6

 

-2.6%

-6.5%

0/3

 

 

±1.6

±1.3

±1.7

 

 

 

 

1000 mg/kg

M

32.5

32.2

31.3

 

-0.9%

-3.7%

0/3

 

 

±0.4

±0.6

±0.8

 

 

 

 

 

F

23.7

23.6

21.8

 

-0.4%

-8.0%

0/3

 

 

±0.6

±0.7

±1.3

 

 

 

 

2000 mg/kg

M

32.5

31.7

29.8

 

-2.5%

-8.3%

1/3

 

 

±2.1

±4.0

±3.0

 

 

 

 

 

F

22.2

19.3

ND

 

-13.1%

ND

3/3

 

 

±1.1

3SD

ND

 

 

 

 

 

1% Change = (Post-treatment weight - Pretreatment weight) x 100

Pretreatment weight

2Reported as number of animals found dead after dose administration/total number tested.

3SD=Standard deviation not available due to only one surviving animal ND=No data due to mortality

M=Male mice, F=Female mice

 

 

Table 2: Toxicity Study - Body Weight and Mortality Data Following Dose Administration of Test item ICR Mice

Group Mean BodyWeights(g)                  %Change¹

Treatment (10 mL/kg)

Sex

Pretreat

Day 1

Day 2

 

Day 1

Day 2

Mortality²

Test item

1200 mg/kg

 

M

 

31.2

 

31.1

 

30.8

 

 

-0.3%

 

-1.3%

 

0/3

 

 

±0.7

±0.5

±0.9

 

 

 

 

 

F

22.7

±1.3

22.1

±0.6

22.4

±1.2

 

-2.6%

-1.3%

1/3

1500 mg/kg

M

33.1

±1.4

32.6

±2.3

32.4

±1.3

 

-1.5%

-2.1%

1/3

 

F

23.9

±1.3

23.4

±0.8

23.3

±0.5

 

-2.1%

-2.5%

1/3

1800 mg/kg

M

32.0

±1.3

NDND

NDND

 

ND

ND

3/3

 

F

25.6

ND

ND

 

ND

ND

3/3

 

 

±2.7

ND

ND

 

 

 

 


1% Change = (Post-treatment weight - Pretreatment weight) x 100

Pretreatment weight

2Reported as number of animals found dead after dose administration/total number tested. ND=No data due to mortality

M=Male mice, F=Female mice

 

 

Table 3: Summary of Bone Marrow Micronucleus Analysis Following Dose Administration of Test Item in ICR Male Mice



 

Treatment           Time(h)  Number    PCE/Total    Control (%)  Number /1000PCEs      Number per PCEs Scored¹ %


PEG 400

 

M

24

5

0.526

±

0.05

---

0.4

±

0.42

4

/

10000

0.04

Test item

250mg/kg        M

 

24

 

5

 

0.492

 

±

 

0.03

 

-6

 

0.5

 

±

 

0.50

 

5

 

/

 

10000

 

0.05

500mg/kg        M

24

5

0.432

±

0.02

-18

0.5

±

0.35

5

/

10000

0.05

1000mg/kg       M

24

5

0.452

±

0.07

-14

0.4

±

0.22

4

/

10000

0.04

 

Cyclophosphamide

 

 

 

 

 

 

 

 

 

 

 

 

 

50 mg/kg

M

24

5

0.346

±

0.04

-34

15.9

±

2.88

*159

/

10000

*1.59

PEG 400

 

 

M

 

 

48

 

 

5

 

 

0.473

 

 

±

 

 

0.07

 

 

--

 

 

0.4

 

 

±

 

 

0.22

 

 

4

 

 

/

 

 

10000

 

 

0.04

Test item

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1000 mg/kg

M

48

5

0.475

±

0.05

0

0.6

±

0.22

6

/

10000

0.06

 

1*Statistically significant, p=<0.05 (Dunnett’st- test) M=Male mice

 

Applicant's summary and conclusion

Conclusions:
Under the conditions of the assay described in this report, a single oral administration of HC Yellow No. 2, at doses up to 1000 mg/kg, did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male ICR mice.
Executive summary:

The purpose of this GLP-compliant study was to evaluate the potential aneugenic and clastogenic effects of the test item HC Yellow No. 2 on ICR mice in In Vivo Micronucleus Assay according to OECD guideline 474 method.

Test concentrations were based on the findings of two dose range finding. In both the pilot toxicity study (3 mice/sex/group treated with 250, 500, 1000 and 2000 mg/kg bw), the toxicity study (3 mice/sex/group treated with 1200, 1500 and 1800 mg/kg bw) and the definitive micronucleus test the study animals were observed for clinical signs of toxicity and mortality immediately after dose administration, approximately 1 h and 4 h post dosing and daily during the course of the experiments. In the definitive experiment mice were exposed by oral gavage to doses of 0, 250, 500 and 1000 mg/kg bw. Bone marrow cells were collected 24 h or 48 h (control and high dose only) after dosing. Toxicity and thus exposure of the target cells was determined by measuring the ratio between polychromatic and total erythrocytes (PCE/EC).

Additionally, satellite groups of 3 mice/sex/time point were treated with the highest dose of test substance (1000 mg/kg bw) for the determination of plasma concentrations of GTS03975. Satellite animals were bled at 1, 2, 4, 6 and 8 h post-dose.However, blood and plasma samples were collected at the testing facility, but plasma samples were not analyzed for the test article concentrations.

In the pilot toxicity study, all animals (treatments of 250, 500, 1000 and 2000 mg/kg bw) showed orange urine, suggesting bioavailability of each applied dose. From the 2000 mg/kg bw group 1 of the 3 males and all 3 females died. From the toxicity study (treatments of 1200, 1500 and 1800 mg/kg bw) 1 of the 3 females of the 1200 mg/kg bw group, 1 male and 1 female of the 1500 mg/kg bw group and all animals of the 1800 mg/kg bw group died. All surviving animals showed orange coloured urine, lethargy, piloerection, orange skin tone.

In the main study, reductions up to 18% in the ratio of PCEs to total erythrocytes were observed in the Test item -treated groups relative to the vehicle control. Biological relevant increases in the number of micronucleated PCEs compared to the concurrent vehicle controls were not found at any dose tested, neither 24 or 48 h after treatment and neither for male and females.

Under the conditions of the assay described in this report, a single oral administration of HC Yellow No. 2, at doses up to 1000 mg/kg, did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, the test substance was concluded to be negative in the micronucleus test using male ICR mice.