Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
01 April 1998 to 04 August 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
OECD Guideline study performed according to GLP. The study was fully reliable (Klimisch score = 1), however the reliability score was lowered to 2 which is the maximum score for read-across. The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Methyl 3-oxo-2-pentylcyclopentaneacetate
EC Number:
246-495-9
EC Name:
Methyl 3-oxo-2-pentylcyclopentaneacetate
Cas Number:
24851-98-7
IUPAC Name:
methyl (3-oxo-2-pentylcyclopentyl)acetate
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): ST 17 C 97
- Substance type: pure active substance
- Physical state: clear colourless liquid
- Expiration date of the lot/batch: 15 Oct 1998
- Storage condition of test material: room temperature protected from light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD - USA
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Pilot study Males: 26.7 - 31.4 g, Females: 25.4 - 27.4 g. Toxicity study: Males: 28.2 - 31.4 g, Females: 24.9 - 28.9 g
Micronucleus assay: Males: 26.1 - 29.9 g Females: 21.5 - 26.7 g
- Assigned to test groups randomly: yes, under following basis: computer-generated program which is based on distribution according to body weight
- Housing: mouse of same sex were housed up to 5 per cage (plastic autoclavable cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding).
- Diet (e.g. ad libitum): ad libitum, certified laboratory rodent chow which had been analysed for environmental contaminants (Harlan TEKLAD certified Rodent 7012C)
- Water (e.g. ad libitum): ad libitum, tap water (Washington Suburban Sanitary Commission, Potomac Plant). The water used in the study met USEPA drinking water standards and is monitored at least annually for levels of organophosphorus pesticides, metals, and coliform and other contaminants.
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
The mice were housed in an AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care International)-accredited facility
- Temperature (°C): 23 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): no data (in accordance with AAALAC.)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: Solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals.
- Amount of vehicle (if gavage or dermal): 20 ml/kg
- Lot/batch no. (if required): corn oil CAS n°8001-30-1 obtained from Super G.
Details on exposure:
Intraperitoneal injection
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by intraperitoneal injection at a constant volume of 20 mL/kg body weight. Intraperitoneal injection was selected to maximize delivery of the test article to the test system. All mice in the experimental and control groups were weighed immediately prior to dose administration, and the dose volume was based on individual body weights.
Duration of treatment / exposure:
Unique treatment by IP injection, bone marrow cells were collected 24 and 48 hours after treatment
Frequency of treatment:
Unique treatment
Post exposure period:
24 and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 280, 560 & 1120 mg/kg bw
Basis:
other: dose injected (intraperitoneal injection)
No. of animals per sex per dose:
At least 5 males and 5 females. See Animal assignment and doses in "Any other information on material and methods including tables"
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CAS n° 6055-19-2, Sigma Chemical Company). Pre-dissolved in sterile distilled H20 at 2.5 mg/ml.
- Justification for choice of positive control(s): Positive control recommended by OECD TG 474
- Route of administration: Intraperitoneal (constant volume of 20ml/kg in corn oil)
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
Bone marrow cells; the incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary pilot study, ST 17 C 97 was administered by intraperitoneal injection to male mice at 1, 10, 100, or 1000 mg/kg bw and to male and female mice at 2000 mg/kg which was administered in a total volume of 20 mL test article-vehicle
mixture/kg bw. Mortality occurred within two days of dose administration as follows: 5/5 males and 5/5 females at 2000 mg/kg. Clinical signs, which were noted following dose administration, included: lethargy in male mice at 1000 mg/kg and prostration, convulsions and irregular breathing in male and female mice at 2000 mg/kg. All other animals appeared normal throughout the observation period.
A toxicity study was then performed; ST 17 C 97 was administered by intraperitoneal injection to male and female mice at 1200, 1400, 1600, or 1800 mg test article/kg bw which was administered in a total volume of 20 mL test article-vehicle mixture/kg bw. Mortality occurred within three days of dose administration as follows: 3/5 males and 4/5 females at 1400 mglkg and all male and female mice at 1600 and 1800 mg/kg. Clinical signs, which were noted following dose administration, included: lethargy in male and female mice at 1200 mg/kg, prostration in one male mouse at 1200 mg/kg and in all male and female mice at 1400 and 1600 and 1800 mglkg. On the days following dose administration signs of lethargy in male mice and one female mouse at 1400 mg/kg, crusty eyes in female mice at 1400 mg/kg and irregular breathing in female mice at 1400 and in one male mouse at 1600 and 1800 mg/kg, were noted. The LD50 was calculated by probit analysis to be approximately 1397.2 mg/kg for male mice and female mice. The high dose for the micronucleus test was set at 1120 mg/kg for male and female mice which was estimated to be approximately 80% of the LD50.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The test article-vehicle mixture, the vehicle alone, or the positive control was administered by intraperitoneal injection at a constant volume of 20 mL/kg body weight. All mice in the experimental aod control groups were weighed immediately prior to dose administration, aod the dose volume was based on individual body weights. Mice were observed after dose administration for clinical signs of chemical effect. Bone marrow was isolated 24 or 48 hours after injection.


DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, up to five mice per sex per treatment were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were 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 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 pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each mouse. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.


METHOD OF ANALYSIS: Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.
In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.
All conclusions were based on sound scientific judgement; 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, Kastenbaum-Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.
Criteria for a Valid Test: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control. The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p ≤ 0.05, Kastenbaum-Bowman Tables).
Statistics:
Described in "Evaluation Criteria" above

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lethargy (m+f) mice at 560 and 1120 mg/kg, and piloerection and prostration in m+f at 1120 mg/kg. Mortality was observed in 4/15 male and 1/15 female receiving 1120 mg/kg.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Solubility: test substance was soluble in corn oil at 100 mg/ml
In a preliminary pilot study, ST 17 C 97 was administered by intraperitoneal injection to male mice at 1, 10, 100, or 1000 mg/kg bw and to male and female mice at 2000 mg/kg which was administered in a total volume of 20 mL test article-vehicle mixture/kg bw. Mortality occurred within two days of dose administration as follows: 5/5 males and 5/5 females at 2000 mg/kg. Clinical signs, which were noted following dose administration, included: lethargy in male mice at 1000 mg/kg and prostration, convulsions and irregular breathing in male and female mice at 2000 mg/kg. All other animals appeared normal throughout the observation period.
A toxicity study was then performed; ST 17 C 97 was administered by intraperitoneal injection to male and female mice at 1200, 1400, 1600, or 1800 mg test article/kg bw which was administered in a total volume of 20 mL test article-vehicle mixture/kg bw. Mortality occurred within three days of dose administration as follows: 3/5 males and 4/5 females at 1400 mglkg and all male and female mice at 1600 and 1800 mg/kg. Clinical signs, which were noted following dose administration, included: lethargy in male and female mice at 1200 mg/kg, prostration in one male mouse at 1200 mg/kg and in all male and female mice at 1400 and 1600 and 1800 mglkg. On the days following dose administration signs of lethargy in male mice and one female mouse at 1400 mg/kg, crusty eyes in female mice at 1400 mg/kg and irregular breathing in female mice at 1400 and in one male mouse at 1600 and 1800 mg/kg, were noted. The LD50 was calculated by probit analysis to be approximately 1397.2 mg/kg for male mice and female mice. The high dose for the micronucleus test was set at 1120 mg/kg for male and female mice which was estimated to be approximately 80% of the LD50.

RESULTS OF DEFINITIVE STUDY (See Table 2 in Remarks on results including table)
- Induction of micronuclei (for Micronucleus assay) & statistical evaluation: The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article treated groups was not statistically increased relative to their respective vehicle control in either male or female mice, regardless of dose level or bone marrow collection time (p > 0.05, Kastenbaum-Bowman Tables). CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p ≤ 0.05, Kastenbaum-Bowman Tables).
- Ratio of PCE/NCE (for Micronucleus assay): Slight reductions of 2 to 12 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to their respective vehicle controls.
- Appropriateness of dose levels and route: doses levels were chosen according to preliminary study to cover range from maximum to little or no toxicity; intraperitoneal route is a route recommended for such assay and recommended in OECD TG 474.



Any other information on results incl. tables

Table 1: Clinical signs following dose administration of ST 17 C 97

Treatment

Clinical Observations

Number of Animals with clinical Signs/Total Number of Animals Dosed

Number of Animals Died/Total Number of Animals Dosed

Males

Females

Males

Females

Corn Oil

20ml/kg

Normal

*/10

*/10

0/10

0/10

ST 17 C 97

280 mg/kg

Normal

*/5

*/5

0/5

0/5

ST 17 C 97

560 mg/kg

Lethargy

3/5

3/5

0/5

0/5

ST 17 C 97

1120 mg/kg

Lethargy

Piloerection

Prostration

13/15

10/15

5/15

15/15

13/15

2/15

4/15

1/15

CP

50 mg/kg

Normal

*/5

*/5

0/5

0/5

 

*= no clinical signs observed

 

Table 2: Summary of Bone Marrow Micronucleus Study

Treatment

Dose (mg/kg)

Sex

Time (h)

Number of mice

PCE/Total Erythrocytes (Mean±SD)

Change From Control (%)

MicronucleatedPolychromatic Erythrocytes

Number per 1000 PCEs

(Mean±SD)

Number per 10000 PCEs scored1

Corn oil

20 ml/kg

0

M

F

24

24

5

5

0.50± 0.06

0.47 ± 0.04

-

-

0.3± 0.27

0.6 ± 0.22

3

6

Test substance (ST 17 C 07)

280

M

F

24

24

5

5

0.44± 0.05

0.46 ± 0.04

-12

-2

0.2± 0.27

0.4 ±0.42

2

4

560

M

F

24

24

5

5

0.45± 0.07

0.48 ± 0.05

-10

2

0.1 ± 0.22

0.2± 0.27

1

2

1120

M

F

24

24

5

5

0.45± 0.07

0.47 ± 0.04

-10

0

0.4 ± 0.42

0.1± 0.22

4

1

CP

50

M

F

24

24

5

5

0.45± 0.09

0.47 ± 0.09

-10

-4

24.6±5.38

22.4 ± 4.38

*246

*224

 

Corn oil

20 ml/kg

0

M

F

48

48

5

5

0.49±0.12

0.54 ± 0.04

-

-

0.2±0.27

0.2 ± 0.45

2

2

Test substance (ST 17 C 07)

1120

M

F

48

48

5

5

0.50± 0.03

0.48 ± 0.05

2

-11

0.8± 0.57

0.4 ± 0.42

8

4

 

1*p0.05 (Kastenbaum-Bowman Tables)

 

Complete data results (individual n° of micronucleated PCE) are attached in "Background attached material "

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The results of the assay indicate that under the conditions described in this report, ST 17 C 97 did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. ST 17 C 97 was concluded to be negative in the mouse micronucleus assay. The test substance is considered as non-mutagenic according to the OECD TG 474: Mammalian Erythrocyte Micronucleus Test.
Executive summary:

Introduction. The test article, ST 17 C 97, was tested in the mouse micronucleus assay according to OECD TG 474 Mammalian Erythrocyte Micronucleus Test. The purpose of this study was to assess the clastogenic potential of a test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice.

 

Methods. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot assay followed by a toxicity study. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 ml/kg bw by a single intraperitoneal injection.

Corn oil was determined to be the solvent of choice based on a solubility determination of the test article and compatibility of the vehicle with the test system animals. The test article was soluble in com oil at 100 mg/ml, the maximum concentration tested. Dosing concentrations were delivered to the test system as solutions.

 

Results. In the pilot assay, male mice were dosed with 1, 10, 100, or 1000 mg test article/kg bw and male and female mice were dosed with 2000 mg/kg. Mortality was observed in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy in male mice at 1000 mg/kg and prostration, irregular breathing and convulsions in male and female mice at 2000 mg/kg.

 

In the toxicity assay, male and female mice were dosed with 1200, 1400, 1600, or 1800 mg test article/kg body weight. Mortality was observed in 3/5 male mice and 4/5 female mice at 1400 mg/kg and in all male and female mice at 1600 and 1800 mg/kg. Clinical signs following dose administration included lethargy in male and female mice at 1200 and 1400 mg/kg, prostration in one male mouse at 1200 mg/kg and in all male and female mice at 1400, 1600 and 1800 mg/kg. Convulsions were noted in all male and female mice at 1400, 1600 and 1800 mg/kg, crusty eyes in female mice at 1400 mg/kg and irregular breathing in female mice at 1400 mg/kg and in one male mouse at 1600 and 1800 mg/kg. The high dose for the micronucleus test was set at 1120 mg/kg which was estimated to be approximately 80% of the LD50.

 

In the micronucleus assay, male and female mice were dosed with 280, 560 or 1120 mg test article/kg body weight. Mortality was observed in 4/15 male and 1/15 female mice dosed with 1120 mg/kg. Clinical signs following dose administration included: lethargy in male and female mice at 560 and 1120 mg/kg, piloerection and prostration in male and female mice at 1120 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight reductions (up to 12%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. No significant increase in micronucleated polychromatic erythrocytes in test article-treated groups relative to the respective vehicle control group was observed in male or female mice at 24 or 48 hours after dose administration (p>0.05,Kastenbaum-Bowman). Cyclophosphamide (positive control) induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p<0.05, Kastenbaum-Bowman).

 

Conclusions. The results of the assay indicate that under the conditions described in this report, ST 17 C 97 did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. ST 17 C 97 was concluded to be negative in the mouse micronucleus assay. The test substance is considered as non-mutagenic according to the OECD TG 474 Mammalian Erythrocyte Micronucleus Test.

The supporting substance is considered adequate for read-across purpose as data relates to a mixture of cis- and trans-isomers whereas the registered substance is the pure cis-isomer (see Iuclid section 13 for additional justification).

Categories Display