Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2014 to 03 August 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
not applicable
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes
Type of assay:
other: damage to chromosomes and/or aneuploidy

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance/physical state: Clear colourless liquid
- Storage conditions: Room temperature in the dark

Test animals

Species:
mouse
Strain:
ICR
Remarks:
CD-1 albino
Sex:
male
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- Sufficient albino Hsd: ICR (CD-1) strain mice were obtained from Harlan Laboratories UK Ltd, Oxon, UK.
- At the start of the main test the mice weighed 22.7 to 30.0 g and were approximately six to ten weeks old.
- After a minimum acclimatisation period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a colour coded cage card.
- Animals were housed in groups of up to seven in solid-floor polypropylene cages with wood-flake bedding.
- Free access to mains drinking water and food (Harlan Teklad 2014C Global Certified Rodent Diet supplied by Harlan Laboratories UK Ltd, Oxon, UK) was allowed throughout the study.
- Representative analyses of food and water quality were retained in the laboratory archive.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- Rate of air exchange was approximately 15 changes per hour.
- Lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
TEST ITEM PREPARATION
- The test item was freshly prepared as required as a solution of appropriate concentration in arachis oil.
- No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
- The test item was formulated within two hours of it being applied to the test system and it was assumed that the formulation was stable for that duration.

POSITIVE CONTROL ITEM
- The positive control item was freshly prepared as required as a solution at the appropriate concentration in distilled water (Laboratoire Aguettant 3010081).

RANGE-FINDING TOXICITY TEST
- A range-finding toxicity test was performed to determine a suitable dose level and route of administration for the main test. The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.
- The range-finding toxicity test was also used to determine if the main test should be performed using both sexes or males only.
- Groups of mice were dosed orally as shown in the table below.
- All animals were dosed once only at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its body weight at the time of dosing.
- Where applicable, animals were observed within the time points of 1 and 4 hours after dosing and subsequently once daily for two days.

MICRONUCLEUS TEST
- Groups of seven mice were dosed once only via the oral route with the test item at 250, 500 or 1000 mg/kg.
- Once group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group, dosed with the test item at 1000 mg/kg, was killed after 48 hours.
- One group of seven mice was dosed via the oral route with vehicle alone (arachis oil) and killed 24 hours after dosing.
- One group of five mice was dosed orally with cyclophosphamide and killed 24 hours after dosing. Cyclophosphamide is a positive control item known to produce micronuclei under the conditions of the test.
- Experimental design is summarised in the table below.
- All animals were observed 1 and 4 hours after dosing and then once daily, as applicable, and immediately prior to termination.
Duration of treatment / exposure:
24 or 48 hours
Frequency of treatment:
Single dose
Post exposure period:
Not applicable
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Seven
Control animals:
yes, concurrent vehicle
Positive control(s):
POSITIVE CONTROL ITEM
- Identification: Cyclophosphamide
- Source: Acros Organics
- Lot number: A0302605
- Harlan serial number: R-5819
- Purity: 97 %
- Expiry date: 06 January 2016
- Storage conditions: Approximately 4 °C in the dark

Examinations

Tissues and cell types examined:
Both femurs were dissected from each animal immediately following sacrifice.
Details of tissue and slide preparation:
SLIDE PREPARATION
- Immediately following termination (24 or 48 hours following dosing), both femurs were dissected from each animal, and aspirated with foetal bovine serum.
- Bone marrow smears were prepared following centrifugation and re-suspension.
- The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa and allowed to air-dry. A cover slip was then applied using mouting medium.
Evaluation criteria:
SLIDE EVALUATION
- 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.

INTERPRETATION OF RESULTS
- A comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the 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 the vehicle control group.
- If these criteria were not fulfilled, the test item 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 vehicle control group.
Statistics:
- 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).
- Data was analysed following a square route (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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY TEST
- Mortality data are summarised in the table below.
- Clinical signs observed in animals dosed with the test item at 2000 mg/kg via the oral route were hunched posture, ptosis, lethargy, ataxia, decreased respiration, laboured respiration and prostration. Due to the severity of the clinical signs, one of the animals was killed in extremis.
- In animals dosed with the test item at 1200 mg/kg via the oral route, clinical signs observed were hunched posture, ptosis, lethargy, ataxia and splayed gait.
- In animals dosed with the test item at 1000 mg/kg via the oral route, clinical signs observed were hunched posture, ptosis, lethargy and tip-toe gait.
- Confirmatory animals were dosed with the test item at 1000 mg/kg and clinical signs of hunched posture, ptosis, ataxia and tip-toe gait were observed.
- The test item showed no marked difference in its toxicity to male or female mice and it was therefore considered acceptable to use males only for the main test.
- With evidence of acceptable toxicity via the oral route, the considered maximum tolerated dose (MTD) of the test item (1000 mg/kg) was selected for use in the main test with 500 mg/kg and 250 mg/kg as the lower dose levels.

MICRONUCLEUS TEST
- There were no premature deaths seen in any of the dose groups.
- Clinical signs of hunched posture, ptosis, ataxia, lethargy and splayed gait were observed at 1000 mg/kg in the 24 and 48-hour dose groups.

EVALUATION OF BONE MARROW SLIDES
- A summary of the results of the micronucleus test is given in Table 1 (attached).
- Individual and group mean data are presented in Tables 2 to 7 (attached).
- There were no statistically significant decreases in the PCE/NCE ratio in any test item exposure group.
- There were no statistically significant increases in the frequency of micronucleated PCEs in the 48-hour 1000 mg/kg dose group or the 250 mg/kg 24-hour 250 mg/kg dose group. Modest but statistically significant increases were observed in the 24-hour 1000 mg/kg and 500 mg/kg dose groups when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean values were well within the historical control range. The responses were therefore considered to be artefactual 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.

Any other information on results incl. tables

MORTALITY DURING RANGE-FINDING TOXICITY TEST

Dose level (mg/kg)

Sex

Number of animals treated

Route

Deaths on Day 0

Deaths on Day 1

Deaths on Day 2

2000

Male

1

Oral

1*

-

-

2000

Female

1

Oral

0

0

0

1200

Male

1

Oral

0

0

0

1200

Female

1

Oral

0

0

0

1000

Male

1

Oral

0

0

0

1000

Female

1

Oral

0

0

0

1000

Male

2

Oral

0

0

0

* = Animal killed in extremis

- = No data

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-genotoxic under the conditions of the test.
Executive summary:

GUIDELINE

The study was performed to assess the potential of the test item to produce damage to chromosomes or aneuploidy when administered to mice. The method was designed to be compatible with the 1997 OECD Guidelines for Testing of Chemicals No 474 “Mammalian Erythrocyte Micronucleus Test”, Method B.12 of Commission Regulation (EC) No 440/2008 of 30 May 2008, the US EPA (TSCA) OPPTS 870.5395, EPA 712-C-98-226, August 1998 guidelines, and be acceptable to the Japanese METI/MHLW/MAFF guidelines for testing of new chemical substances.

 

METHODS

A range-finding test was performed to identify suitable dose levels of the test item, route of administration and to investigate whether there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity between the sexes and the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven male mice at the maximum tolerated dose of 1000 mg/kg with 500 mg/kg and 250 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. Additional groups of mice were given a single oral dose of arachis oil (7 mice) or doses orally with cyclophosphamide (5 mice) to serve as vehicle and positive controls respectively. Vehicle and positive control animals were killed 24 hours later.

 

RESULTS

There were no premature deaths in any of the dose groups. Clinical signs of hunched posture, ptosis, ataxia, lethargy and splayed gait were observed at 1000 mg/kg in the 24 and 48-hour dose groups. No statistically significant decreases in the PCE/NCE ratio were observed in any test item dose groups when compared with the vehicle control group. There were no statistically significant increases in the frequency of micronucleated PCEs in the 48-hour 1000 mg/kg group or the 24-hour 250 mg/kg group. Modest but statistically significant increases were observed in the 24-hour 1000 mg/kg and 500 mg/kg groups when compared to the vehicle control group. However, the group mean for the vehicle control was very low, none of the individual animals had any marked increases in the number of micronucleated PCE, and the group mean values were well within the historical control range. The responses were therefore considered to be artefactual 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 know mutagenic activity of cyclophosphamide under the conditions of the test.

 

CONCLUSION

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