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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:
09 July 1984 to 17 November 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
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 the relevant results. The study report was conclusive, done to a valid guidelines and the study was conducted under GLP conditions. The study was performed on a structurally similar substance the results of which are read-across and considered to be representative of the effects of the registered substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report Date:
1984

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): salicylic acid cyclohexyl ester
- Physical state: Colourless liquid
- Molecular weight (if other than submission substance): 220.27

Test animals

Species:
mouse
Strain:
other: CFW 1 (Winkelmann)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Approximately 20-30 g
- Assigned to test groups randomly: yes (using a random number table)
- Fasting period before study: Animals in the 3000 mg/kg group were deprived of food for 16 hours prior and 3 hours post dosing. For all other animals, feed was withheld for 4 to 6 hours prior to dosing and 3 hours post dosing.
- Housing: Individually
- Diet: Altromin standard diet no. 1324, 10 mm pellets (Altrogge speciality feed)
- Water: Tap water was available to the animals ad libitum

ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

IN-LIFE DATES: From: 06 August 1984 To: 09 August 1984

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: arachis oil
- Amount of vehicle: 10 mL/kg
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test material was diluted with the carrier immediately before application
Duration of treatment / exposure:
A single oral gavage treatment was administered.
Frequency of treatment:
One single treatmentFurther details of test material (as cited in IUCLID section 1.2):
- A main constituent of the substance defined in section 1
- Concentration range of the constituent in the substance: 10-20%
Post exposure period:
Up to 72 hours
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
Dose / conc.:
3 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
7 animals per sex, per dose in the vehicle control, positive control and 300 and 1500 mg/kg bw of test material.
7 animals per sex per dose per time point were dosed with 3000 mg/kg bw (a total of 21 males and 21 females per dose)
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance used: Cyclophosphamide
- Route of administration: Intraperitoneal
- Dose : 20 mg/kg bw

Examinations

Tissues and cell types examined:
Bone marrow (polychromatic erythrocytes)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A dose range finding toxicity study was performed with male and female mice of the same strain as the definitive test, in which the animals were dosed via oral gavage with the test material in arachis oil at doses of 3000, 4000 and 5000 mg/kg bw. The doses used in the definitive test were selected based on the results of the range finding toxicity study.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
All animals were dosed with a single oral gavage dose of the appropriate treatment, with the exception of the positive control animals who were dosed with a single intraperitoneal injection.
All doses were sampled at 24 hours post dosing. In addition samples were gathered from animals dosed with 3000 mg/kg bw at 48 and 72 hours also.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed at the appropriate time points (24, 48 and 72 hours) using ether. The femurs were extracted and the proximal end of the femur was opened. 0.2 mL of foetal calf serum (FCS) was injected into the medullary end of the cavity. The femur was immersed in FCS and flushed out several times so that the bone marrow bulged out as a fine suspension. The bone marrow was centrifuged at 1000 rpm for five minutes, the supernatant was removed using a siliconised pipette and the pellet was mixed with a new pipette. A small drop of the sediment was deposited onto a degreased microscope slide and spread evenly.
Three smears were prepared per animal. The slides were left to dry overnight and then were stained.
The slides were fixed in absolute methanol (five minutes) then rinsed twice with distilled water and stained with diluted Giemsa solution (1 part Giemsa:1 part glycerin diluted in 5 parts bidistilled water) for five minutes following which the slides were rinsed with distilled water and left to dry naturally overnight.

METHOD OF ANALYSIS:
From the three prepared slides, one preparation was selected for analysis. The number of micronuclei in 1000 polychromatic erythrocytes was counted for each test animal. Slides were assessed at a magnification of 8 x 100-times, a region of each slide was selected based on the quality of staining and spread of cells at medium magnification.
The ratio of polychromatic erythrocytes (stained blue) to normocytes (stained red) was determined.
Evaluation criteria:
The slides were evaluated for a statistically and time-dependent increase in the number of micronucleated polychromatic erythrocytes (PCE).
Statistics:
Kastenbaum and Bowman tables were used to calculate the statistical significance of the test material groups compared to the control.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No deaths occurred in the treatment groups
Vehicle controls validity:
valid
Negative controls validity:
not valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 3000, 4000 and 5000 mg/kg bw
- Clinical signs of toxicity in test animals: A single male died at 4000 mg/kg bw between 0 and 24 hours. One male died in the 5000 mg/kg bw group between 0 and 24 hours, and one male and one female died in the 5000 mg/kg bw group between 24 and 48 hours. In the 3000 mg/kg bw group, slightly reduced activity and accelerated respiration was observed in two animals of each sex. One of the affected females exhibited shaking and uncoordinated gait which had recovered by 24 hours. At 4000 mg/kg bw, two animals of each sex exhibited slightly reduced activity and slightly increased respiration. Both of the affected male mice had ruffled fur. 24 hours after administration, one of these males was found dead. The second male mouse was found to still have ruffled fur. The toxicity exhibited by the females had subsided by


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No statistically significant increase in micronucleated polychromatic erythrocytes at the highest dose tested.
- Ratio of PCE/NCE (for Micronucleus assay): No influence on PCE/NCE ratio.
- Statistical evaluation: A statistically and time-dependent increase was not observed in either sex.

Any other information on results incl. tables

Table 1: Results

Treatment Group

(mg/kg bodyweight)

Sex

Time of Sacrifice

(hours)

Mortality of test animals

Micronuclei/1000 PCE

Ratio of Polychromatic /Normochromatic Erythrocytes

Mean

Range

Mean

Range

Vehicle Control

(10 mL/kg Arachis oil)

M

24

0/7

0.71

0-1

1.09

0.84-1.57

F

24

0/7

0.71

0-1

1.52

1.41-1.69

Positive control

(Cyclophosphamide 20 mg/kg bw)

M

24

0/7

12.90

10-15

1.14

0.90-1.32

F

24

0/7

11.60

8-20

1.08

0.76-1.22

300

M

24

0/7

*

*

*

*

F

24

0/7

*

*

*

*

1500

M

24

0/7

*

*

*

*

F

24

0/7

*

*

*

*

3000

M

24

0/7

1.43

0-3

1.09

0.88-1.46

F

24

0/7

1.00

0-2

1.38

1.03-1.88

M

48

0/7

1.00

0-3

0.74

0.32-1.10

F

48

0/7

0.86

0-2

1.14

0.59-1.52

M

72

0/7

0.86

0-2

0.80

0.44-1.24

F

72

0/7

1.43

0-3

1.34

1.00-1.71

*Not evaluated

Applicant's summary and conclusion

Conclusions:
Interpretation of results: negative
Under the conditions of the test, the test material did not induce a statistically or a time dependent increase in micronucleated polychromatic erythrocytes in male and female mice when tested up to concentrations of 3000 mg/kg.
Executive summary:

The cytogenicity of the test material in vivo was investigated in a GLP compliant study conducted in accordance with OECD 474. The test material, cyclohexyl salicylate was administered by oral gavage in arachis oil at doses up to 3000 mg/kg in male and female mice. The animals were sacrificed at 24, 48 and 72 hours, and smears were prepared from the bone marrow. Under the conditions of the test, the test material did not induce a statistically or a time dependent increase in micronucleated polychromatic erythrocytes in male and female mice when tested up to concentrations of 3000 mg/kg.