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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between July 17, 2000 and August 10, 2000.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
This study was performed in accordance with Good Laboratory Practice standards. The study followed the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report, Part 1 revised (Basic Mutagenicity Tests: UKEMS recommended procedures, 1990). The study design also complied with the revised OECD Guidelines for Testing of Chemicals Number 474 Micronucleus Test and Method B.12 of the EEC Commission Directive 92/69/EEC.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

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
Qualifier:
according to
Guideline:
other: Basic Mutagenicity Tests: UKEMS recommended procedures, 1990
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LZ1224.20; Diethyltoluenediamine; DETDA 80
- Physical state: Amber slightly viscous liquid
- Analytical purity: 98.4%
- Impurities (identity and concentrations): 0.4% ethyl toluenediamine (GC); <0.0150% toluenediamine (GC); 0.02% (w/w) water (KF)
- Composition of test material, percentage of components: 77.6% 3,5-diethyltoluene-2,4-diamine (GC); 20.8% 3,5-diethyltoluene-2,6-diamine (GC)
- Purity test date: August 31, 2000
- Lot/batch No.: 2235
- Storage condition of test material: Approximately 4 degrees Celsius under nitrogen

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent
- Age at study initiation: Approximately 5 to 8 weeks old
- Weight at study initiation: 25 to 30 grams
- Assigned to test groups randomly: [yes, under following basis: animals were selected at random and given a number unique within the study by ear punching and a number on a color coded cage card]
- Fasting period before study:
- Housing: Groups of up to 7 in solid-floor polypylene cages with woodflake bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 15 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light


IN-LIFE DATES: From: July 5, 2000 To: July 7, 2000

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was freshly prepared as required as a solution at the appropriate concentration in arachis oil.
Duration of treatment / exposure:
All animals were dosed once only at the appropriate dose level by gavage.
Post exposure period:
Animals were observed for signs of overt toxicity and death one hour after dosing and then once daily as applicable. For the preliminary range-finding study, animals were sacrificed at 48 hours following dosing. For the micronucleus study, animals were sacrificed at 24 or 48 hours post dosing.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
12.5 mg/ml (125 mg/kg bw)
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
25 mg/ml (250 mg/kg bw)
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
50 mg/ml (500 mg/kg bw)
No. of animals per sex per dose:
7
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): Shown to produce micronuclei under the conditions of the test
- Route of administration: oral gavage
- Doses / concentrations: 50 mg/kg; 5 mg/ml

Examinations

Tissues and cell types examined:
Polychromatic and normochromatic erythrocytes were examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The doses selected for the micronucleus study were based on the results from a range-finding toxicity study in which 2 male and 2 female mice were administered the test material at dose levels of 300 mg/kg, 450 mg/kg, or 600 mg/kg by oral gavage.


TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Groups of mice (n=7/group) were dosed once only via the oral route with the test material at 125 mg/kg, 250 mg/kg, or 500 mg/kg. One group of mice from each dose level was killed by cervical dislocation 24 hours following treatment and a second group dosed at 500 mg/kg at 48 hours. In addition, three further groups of mice were included in the study; two groups (n=7/group) were dosed via the oral route with the vehicle alone (arachis oil) and a third group (n=5) was dosed orally with the positive control material. 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 sacrifice (i.e., 24 or 48 hours following dosing), both femurs were dissected from each animal, aspirated with fetal calf serum and bone marrow prepared following centrifugation and resuspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa, allowed to air-dry and coverslipped 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 occassionally 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 were 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 demonstrated, 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 shwon to be statistically significantly lower than the concurrent vehicle control group.
Statistics:
All data were statistically analyzed using appropriate statistical methods as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data were analyzed following a square root of (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
Remarks:
up to the limit dose of 500 mg/kg bw
Toxicity:
yes
Remarks:
observed at 125 mg/kg bw and higher
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING TOXICITY STUDY
The dose levels for the preliminary study were 300 mg/kg bw, 450 mg/kg bw, and 600 mg/kg bw. In animals dosed with the test material via the oral route, premature death occurred at 600 mg/kg bw and clinical signs were observed at and above 300 mg/kg, as follows: lethargy, hunched posture, ptosis, decreased respiratory rate, labored respiration, ataxia, and splayed gait.

The test material showed no marked difference in its toxicity to male or female mice. Therefore, males were used for the definitive study. Adequate evidence of test material toxicity was demonstrated via the oral route of administration to warrant the same route in the main study. The maximum tolerated dose of the test material, 500 mg/kg bw, was selected for use in the main study, with 250 and 125 mg/kg bw as the lower dose levels.

MICRONUCLEUS STUDY
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 125 mg/kg bw in both the 24 and 48-hour groups, where applicable, these were as follows: hunched posture, lethargy, and ptosis.

Evaluation of Bone Marrow Slides
There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. However, the presence of clinical observations indicated that systemic absorption had occurred. There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.

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 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 did not produce an increase in the frequency of micronuclei in polychromatic erythrocytes, and was therefore considered non-genotoxic under the conditions of this test.