Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2009 to 30 September 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Date of inspection: 19 August 2008; date of signature: 04 March 2009
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : H-CB sodium salt
Description : blue solid
Chemical name : Copper phthalocyanine direct dyes
Purity : 94.8%
Lot number : MB-2
Label : Cyan H-CB Lot: MB-2 Net:1kg
Date received : 04 December 2008
Storage conditions : room temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK
- Age at study initiation: five to eight weeks old
- Weight at study initiation: 22 to 30g
- Assigned to test groups randomly: yes
- Fasting period before study: None
- Housing: solid-floor polypropylene cages with wood-flake bedding
- Diet (e.g. ad libitum): Free access to food (Harlan Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains drinking water was allowed throughout the study.
- Acclimation period: Minimum acclimatisation period of five days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours light and twelve hours darkness

IN-LIFE DATES: From: start of study To: termination

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil for 2000 mg/kg dose level; distilled water for educed concentrations
- Justification for choice of solvent/vehicle: The vehicle was changed to distilled water as the test material was considered to be soluble at these reduced concentrations and more appropriate for the intraperitoneal route of administration.
- Concentration of test material in vehicle: 5, 10 and 20 mg/ml
- Amount of vehicle (if gavage or dermal): > 10ml/kg
Duration of treatment / exposure:
One dose only.
Frequency of treatment:
Once only.
Post exposure period:
Animals were observed 1 hour after dosing and then once daily as applicable and immediately prior to termination. Three groups (50, 100 and 200 mg/kg respectively) were observed for 24 hours in total whilst a fourth group (200 mg/kg) was observed for 48 hours.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
nominal conc.
Intraperitoneal
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
nominal conc.
Intraperitoneal
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
nominal conc.
Intraperitoneal
No. of animals per sex per dose:
Range Finding Study
2000 mg/kg: one male, one female
1000 mg/kg: one male, one female
500 mg/kg: one male, one female
200 mg/kg: three males, one female

Main Study
200 mg/kg: fourteen male
100 mg/kg: seven male
50 mg/kg: seven male
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): Cyclophosphamide is a positive control material known to produce micronuclei under the conditions of the test.
- Route of administration: Oral
- Doses / concentrations: one dose of 50 mg/kg

Examinations

Tissues and cell types examined:
frequency of micronucleated polychromatic (PCE) erythrocytes and normochromatic (NCE) erythrocytes
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Groups, each of seven mice, were dosed once only via the intraperitoneal route with the test material at 200, 100 or 50 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed with test material at 200 mg/kg was killed after 48 hours. In addition, three further groups of mice were included in the study; two groups (seven mice) were dosed via the intraperitoneal route with the vehicle alone (distilled water) and a third group (five mice) was dosed orally with cyclophosphamide. The vehicle controls were killed 24 or 48 hours following dosing and positive control group animals were killed 24 hours following dosing.

DETAILS OF SLIDE PREPARATION: Immediately following termination (i.e. 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS: Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. Micronuclei are normally circular in shape, although occasionally they may be oval or half-moon shaped, and have a sharp contour with even staining. In addition, the number of normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were also scored for incidence of micronuclei.
The ratio of polychromatic to normochromatic erythrocytes was calculated together with appropriate group mean values and standard deviations.
Evaluation criteria:
A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test material groups and the number occurring in the corresponding vehicle control group.
A positive mutagenic response was demonstrated when a statistically significant, dose responsive, toxicologically relevant increase in the number of micronucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
If these criteria were not fulfilled, then the test material was considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the concurrent vehicle control group.
All data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a √(x +1) transformation using Student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 200 - 2000 mg/kg
- Solubility: yes
- Clinical signs of toxicity in test animals: Yes; Hunched posture, ptosis, extremities blue in colour, faeces stained blue, urine stained blue, ataxia, tiptoe gait and diarrhoea.
- Evidence of cytotoxicity in tissue analyzed: Not stated

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): polychromatic erythrocytes
- Ratio of PCE/NCE (for Micronucleus assay):
Group Mean
200 mg/kg 48 hour sampling time: 0.80
200 mg/kg 24 hour sampling time: 0.63*
100 mg/kg 24 hour sampling time: 0.96
50 mg/kg 24 hour sampling time: 0.90
* =P < 0.05

Any other information on results incl. tables

Range-finding Toxicity Test

The mortality data are summarised as follows:

Dose Level

(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

2000

Male

1

oral

0

0

0

0/2

Female

1

0

0

0

1000

Male

1

ip

1

-

-

2/2

Female

1

1

-

-

500

Male

1

ip

1

-

-

2/2

Female

1

1

-

-

200

Male

1

ip

0

0

0

0/2

Female

1

0

0

0

200

Male

2

ip

0

0

0

0/2

Female

0

-

-

-

ip = Intraperitoneal

-  = No data

Insufficient evidence of toxicity was observed in animals dosed with test material via the oral route and, therefore systemic absorption could not be confirmed using this dose route.

In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 500mg/kg, and clinical signs were observed at and above200 mg/kg as follows: Hunched posture, ptosis, extremities blue in colour, faeces stained blue, urine stained blue, ataxia, tiptoe gait and diarrhoea.

The test material showed no marked difference in its toxicity to male or female mice; it was therefore considered to be acceptable to use males only for the main test. Adequate evidence of test material toxicity was demonstrated via the intraperitoneal route of administration; therefore, this was selected for use in the main test. The maximum tolerated dose (MTD) of the test material, 200 mg/kg, was selected for use in the main test, with 100 and 50 mg/kg as the lower dose levels.

Micronucleus Test

Mortality Data and Clinical Observations

There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 50 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ano-genital area stained blue, fur stained blue, extremities blue in colour, faeces stained blue and urine stained blue.

Evaluation of Bone Marrow Slides

A summary of the results of the micronucleus test is given in Table 1 below.

Table1              Micronucleus Test - Summary of Group Mean Data

Treatnt Group

Number of PCE with Micronuclei per 2000 PCE

PCE/NCE Ratio

Group Mean

SD

Group Mean

SD

1.      Vehicle Control
10 ml/kg
48-hour Sampling Ti

0.6

0.8

0.83

0.24

2.      Vehicle Control
10 ml/kg
24-hour Sampling Ti

0.1

0.4

0.85

0.18

3.      Positive Control
50 mg/kg
24-hour Sampling Ti

21.8***

8.2

1.00

0.38

4.      H-CBsodium salt
200 mg/kg
48-hour Sampling Ti

1.6

1.4

0.80

0.27

5.      H-CBsodium salt
200 mg/kg
24-hour Sampling Ti

0.7

1.3

0.63*

0.13

6.      H-CBsodium salt
100 mg/kg
24-hour Sampling Ti

1.3*

1.4

0.96

0.28

7.      H-CBsodium salt
50 mg/kg
24-hour Sampling Ti

1.4*

1.5

0.90

0.31


PCE=Polychromatic erythrocytes

NCE=Normochromatic erythrocytes

SD   =Standard deviation

*        =P < 0.05

***   =P < 0.001

Individual and group mean data are presented in Tables 2 to 8 found in Attachment 1 below.

A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 200 mg/kg test material dose group when compared to the concurrent control groups. This decrease, together with the observation of clinical signs in both the 24 and 48-hour test material dose groups, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no statistically significant increase in the frequency of micronucleated PCE in the 48-hour 200 mg/kg test material dose group. Very modest but statistically increases in the frequency of micronucleated PCE were observed in the 24-hour 100 and 50 mg/kg dose groups. However, there was no evidence of a statistically significant increase in the 24-hour 200 mg/kg dose group, the group means of micronucleated PCE were within the acceptable range for vehicle controls, and the statistical significance was considered to be due to the comparison to the very low concurrent vehicle control group mean. Therefore, the responses were considered to be spurious and of no toxicological significance.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

The test material was found not to produce a toxicologically significant increase in the frequency of micronuclei in polychromatic erythrocytes of mice under the conditions of the test.


Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The test material was considered to be non-genotoxic under the conditions of the test.
Executive summary:

Introduction. The study was performed to assess the potential of the test material to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to comply with the 1997 OECD Guidelines for Testing of Chemicals No.474 "Micronucleus Test", Method B12 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the USA EPA, TSCA and FIFRA guidelines and the Japanese METI/MHLW guidelines for testing of new chemical substances.

Methods. A range-finding test was performed to find suitable dose levels of the test material, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 200 mg/kg and with 100 and 50 mg/kg as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei.

Further groups, each of 7 mice, were given a single intraperitoneal dose of distilled water or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours.

Results. There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test material at and above 50 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ano-genital area stained blue, fur stained blue, extremities blue in colour, faeces stained blue and urine stained blue.

A modest but statistically significant decrease in the PCE/NCE ratio was observed in the 24-hour 200 mg/kg test material dose group when compared to the concurrent control groups. This decrease, together with the observation of clinical signs in both the 24 and 48-hour test material dose groups, was taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no statistically significant increase in the frequency of micronucleated PCE in the 48-hour 200 mg/kg test material dose group. Very modest but statistically increases in the frequency of micronucleated PCE were observed in the 24-hour 100 and 50 mg/kg dose groups. However, there was no evidence of a statistically significant increase in the 24-hour 200 mg/kg dose group, the group means of micronucleated PCE were within the acceptable range for vehicle controls, and the statistical significance was considered to be due to the comparison to the very low concurrent vehicle control group mean. Therefore, the responses were considered to be spurious and of no toxicological significance.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion. The test material was considered to be non-genotoxic under the conditions of the test.