Dimethyl adipate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

This IUCLID for dimethyl adipate is compiled using data from the substance itself, a structuraly related compound (dimethyl glutarate) and a mixture of dibasic esters (dimethyl adipate, succinate and glutarate). The toxicity of each of these susbtances is similar and the document attached justifies why data on the category members can be used to support the data gaps for dimethyl adipate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Dimethyl adipate

EC Number:

211-020-6

EC Name:

Dimethyl adipate

Cas Number:

627-93-0

Molecular formula:

C8H14O4

IUPAC Name:

dimethyl adipate

Details on test material:

- Name of test material (as cited in study report): Dimethyl adipate
- Physical state: clear liquid
- Analytical purity: 98.824%
- Lot/batch No.: J806339-A
- Expiration date of the lot/batch: Sponsor's responsibility
- Stability under test conditions: sufficiently stable for the duration of the study
- Supplied by: DuPont

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: 6-8 weeks at receipt
- Weight at study initiation: males - 151 - 172.4 grams, females - 126.1 - 144.1 grams at receipt
- Assigned to test groups randomly: yes
- Housing: group housed sex wise
- Diet (e.g. ad libitum): Animals were provided with pellet expanded rat and mouse no. 1 maintenance diet (SQC grade obtained from Special Diets Services Ltd., Witham, Essex, UK), withheld during the period of inhalation exposure
- Water (e.g. ad libitum):tap water provided ad libitum, withheld
- Acclimation period: minimum of 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 50 ± 10%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Administration / exposure

Route of administration:

inhalation: aerosol

Vehicle:

- Vehicle(s)/solvent(s) used: none

Details on exposure:

TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Animals were exposed in whole body exposure chambers constructed from stainless steel and glass. The internal volume of each chamber was approximately 750 litres
- Method of holding animals in test chamber: Animals were housed singly in stainless steel mesh compartments during exposure
- Source and rate of air: air was introduced into each exposure chamber at a total rate of 150 litres per minute.
- System of generating particulates/aerosols: The liquid test material was delivered to a concentric jet atomiser and generated as droplets into a stream of dry air for administration to the rats by inhalation while held in whole body exposure chambers. The target concentrations were achieved by metering the test substance from polypropylene syringes mounted on syringe drivers. The atmospheres produced by the atomisers were further diluted with air to give the final chamber concentrations.
- The flow through each chamber was approximately 12 air changes an hour, suffcient to maintain oxygen concentration above 19% v/v, temperature approximately 22 ± 1°C and realtive humidity between 40 - 60%. The exposure chamber was maintained 1-10 mm H2O below ambient pressure

TEST ATMOSPHERE
- Brief description of analytical method used: The samples of chamber air were collected by drawing a previously selected volume of chamber atmosphere through a sintered glass bubbler containing 1,4-dioxane as a trapping agent. Sample collection was performed by inserting the inlet of bubbler through a port located at the animal exposure level on the side wall of the chamber. The atmosphere samples were taken at a claibrated flow of 2 litres/minute using a laboratory pump. The air volume of each sample collected was measured using an in-line wet type gas meter. The mass of the test material collected was determined by gas chromatography (GC) and the aerosol concentration derived from the mass of Dimethyl adipate found and the air volume sampled. Droplet size distribution was evaluated by using marple sampler (impactor stage).
- Samples taken from breathing zone: yes

Duration of treatment / exposure:

two six hour periods on two consecutive days

Frequency of treatment:

two six hour periods on two consecutive days

Post exposure period:

not applicable

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
Air
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0.5 mg/l Dimethyl adipate
Basis:
nominal conc.

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1.0 mg/l Dimethyl adipate
Basis:
nominal conc.

Doses / concentrations 4

Remarks:

Doses / Concentrations:
2.0 mg/l Dimethyl adipate
Basis:
nominal conc.

No. of animals per sex per dose:

5 males + 5 females

Control animals:

yes

Positive control(s):

Cyclophosphamide (Sigma Chemical Co. Ltd., batch number 87H0207) - prepared as a solution in purified water (batch number 00I001B26) at a concentration of 2 mg/l just prior to administration.
- Justification for choice of positive control(s): recommeded positive control by various regulatory agencies
- Route of administration: oral by intragastric gavage using a dose volume of 10 ml/kg and at a time consistent with the end of the second exposure
- Doses / concentrations: 20 mg/kg

Examinations

Tissues and cell types examined:

Observation of micronuclei in erythrocytes of bone marrow smear collected from femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: based on previous studies - preliminary, acute and repeated dose studies.

TREATMENT AND SAMPLING TIMES: 5 males and 5 females were sacrificed from the negative control and each of the test substaance groups 24 hours after completion of the second exposure period. The positive control group was sacrificed 24 hours after dosing.

DETAILS OF SLIDE PREPARATION: - The animals were killed by cervical dislocation following carbon dioxide inhalation and both femurs dissected out from each animal. The femurs were cleared of tissue and the proximal epiphysis and removed from each bone. The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 10 ml Hanks balanced salts solution by aspiration through a 21 gauge needle fitted to a syringe. The resulting cell suspensions were centrifuged at 1000 rpm for 5 minutes and the supernatant discarded. Each resulting cell pellet was resuspended in 2 ml of filtered fetal calf serum before being sedimented by centrifugation. The supernatant was discarded and the final cell pellet was resuspended in a small volume of fetal calf serum to facilitate smearing in the conventional manner on glass microscopic slides. Due to the presence of mast cell granules in rat bone smears, which appear identical to micronuclei a modified Feulgen staining method was used. The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal.

METHOD OF ANALYSIS: The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Also, the proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes.

Evaluation criteria:

A positive response is normally indicated by a statistically significant dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compared with the concurrent control group ( p< 0.01); individual and/or group means exceed the laboratory historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or a negative response.

Statistics:

Standard and recommended statistical procedures were used.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No mortalities were obtained in the micronucleus test.

Clinical signs such as partially closed eyes, smacking mouth, lethargy, piloerection, toe-walking, oily fur, yellow staining in urogenital region, brown staining around snout and wet fur in urogenital region were noted in animals exposed to aerosol of dimethyl adipate. No adverse clinical signs were noted in the negative control group animals. Non-significant incidences of body weight loss were recorded during the test period.

Exposure to dimethyl adipate did not cause any statistically significant increases in the number of micronucleated immature erythrocytes (p > 0.1). Cyclophospahmide administered group caused large highly significant increases in the frequency of micronucleated immature erythrocytes (p < 0.001).

Dimethyl adipate exposed group did not cause any substantial increases in the incidence of micronucleated mature erythrocytes and failed to cause any significant decreases in the proportion of immature erythrocytes (p > 0.01) whicle cyclophosphomide adminsitered group caused statistically significant decreases in the proportion of immature erythrocytes.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Dimethyl adipate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure at the hightest tested dose of 2 mg/l.

Executive summary:

The study was designed to assess the potential induction of micronuclei by Dimethyl adipate in bone marrow cells of F344 rats. Animals were treated for two six hour periods of whole body inhalation exposure, to the test substance at dose levels of 0.5, 1.0 and 2.0 mg/l (expressed as weight of the test substance per unit volume of chamber air).

The test substance and negative control were administered by whole body exposure. The negative control group received clean air only. A positive control group was dosed orally, by gastric intubation, with cyclophosphamide at 20 mg/kg body weight.

No mortalities were obtained in the micronucleus test. Clinical signs such as partially closed eyes, smacking mouth, lethargy, piloerection, toe-walking, oily fur, yellow staining in urogenital region, brown staining around snout and wet fur in urogenital region were noted in animals exposed to aerosol of dimethyl adipate. No adverse clinical signs were noted in the negative control group animals. Non-significant incidences of body weight loss were recorded during the test period.

Bone marrow smears were prepared from 5 male and 5 female animals in the negative control and each of the test groups 24 hours after completion of the second exposure period and from the positive control group 24 hours after dosing. One smear from each animal was examined for the presence of micronuclei in 2000 immature eryhthrocytes. The proportion of immature erythrocytes was assessed by examining of at least 1000 erythrocytes from each animal.

Exposure to dimethyl adipate did not cause any statistically significant increases in the number of micronucleated immature erythrocytes (p > 0.1). Cyclophospahmide administered group caused large highly significant increases in the frequency of micronucleated immature erythrocytes (p < 0.001).

Dimethyl adipate exposed group did not cause any substantial increases in the incidence of micronucleated mature erythrocytes and failed to cause any significant decreases in the proportion of immature erythrocytes (p > 0.01) whicle cyclophosphomide adminsitered group caused statistically significant decreases in the proportion of immature erythrocytes.

Dimethyl adipate did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered by whole body inhalation exposure at the hightest tested dose of 2 mg/l.