Registration Dossier

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:
supporting study
Study period:
July to September 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

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

Materials and methods

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Naphthalene-2,6-dicarboxylic acid
EC Number:
214-527-0
EC Name:
Naphthalene-2,6-dicarboxylic acid
Cas Number:
1141-38-4
Molecular formula:
C12H8O4
IUPAC Name:
naphthalene-2,6-dicarboxylic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Purity: >95%

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Limited, Margate, Kent, England
- Weight at study initiation: Males weighed between 28 and 30 grams and females weighed between 22 and 24 grams.
- Housing: plastic disposable cages
- Diet (e.g. ad libitum): day. All animals were allowed free access to pelleted expanded rat and mouse No. 1 maintenance diet.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 6 to 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C
- Humidity (%): 50%
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours per day

No additional data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: aqueous 1% methylcellulose
- Lot/batch no. (if required): T70654

No additional data
Details on exposure:
2,6-Naphthalenedicarboxylic acid and negative control (aqueous 1% methylcellulose) were dosed by intraperitoneal injection, and mitomycin C was administered orally by intragastric gavage.
Duration of treatment / exposure:
48 hours
Frequency of treatment:
once
Post exposure period:
None stated
Doses / concentrations
Remarks:
Doses / Concentrations:
120, 240 and 480 mg/kg bodyweight
Basis:
nominal conc.
No. of animals per sex per dose:
5 males and 5 females for 120 mg/kg bodyweight, 5 males and 5 females for 240 mg/kg bodyweight, 10+2 males and 10+2 females for 480 mg/kg bodyweight,
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C
- Justification for choice of positive control(s):
- Route of administration: intragastric gavage
- Doses / concentrations: 12 mg/kg bodyweight

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: From the results obtained in the preliminary toxicity study, dose levels of 120, 240 and 480 mg/kg bodyweight were chosen for the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Five males and five females from the negative control, each of 2,6-NDA groups and the positive control group were sacrificed 24 hours after dosing. In addition five male and five female animals in the negative control group and four male and four female animals in the high level treatment group were sacrificed 48 hours after dosing.

DETAILS OF SLIDE PREPARATION: The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 mL of prefiltered foetal calf serum using a 2 mL disposable syringe fitted with a 21 gauge needle. The cells were sedimented by centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner. At least three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa. Following rinsing in distilled water and differentiation in buffered distilled water (pH 6.8), the smears were air-dried and mounted with coverslips using DPX.

No additional data
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 mean values should exceed the laboratory historical control range (Morrison and Ashby 1995). A negative result is indicated where individual and group mean incidences of micronucleated immature erythrocytes for the group treated with 2,6-NDA are not significantly greater than incidences for the concurrent control group (P>0.01) and where these values fall within the historical control range. An equivocal response is obtained when the results do not meet the criteria specified for a positive or negative response.
Statistics:
The results for each treatment group were compared with the results for the concurrent control group using non-parametric statistics.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Three male and three female animals died after treatment with 2,6-NDA at the high dose level.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Phase I: 250, 500, 1000 and 2000 mg/kg bodyweight; Phase II: 500, 600, 720 and 864 mg/kg bodyweight
- Clinical signs of toxicity in test animals: All the animals died at 1000 and 2000 mg/kg bodyweight.

No additional data

Any other information on results incl. tables

Clinical signs and mortalities

Three male and three female animals died after treatment with the test substance at the high dose level. Where possible, these animals were replaced by animals from the concurrently treated satellite group. At post mortem examination, none of the dead animals showed signs of mis-dosing.

The mortalities and clinical signs for the high level group were consistent with the maximum tolerated dose having effectively been achieved. No adverse clinical signs were obtained for the vehicle control or positive control treated animals over the duration of the test.

Micronucleated immature erythrocyte (mie) counts

naphthalene-2,6-dicarboxylic acid did not cause any statistically significant increases in the number of micronucleated immature erythrocytes at either sampling time [P>0.01].

Mitomycin C caused large, highly significant increases [P<0.001] in the frequency of micronucleated immature erythrocytes.

Micronucleated mature erythrocytes (mme)

naphthalene-2,6-dicarboxylic acid did not cause any substantial increases in the incidence of micronucleated mature erythrocytes at either sampling time.

Proportion of immature erythrocytes (% ie/ie + me)

naphthalene-2,6-dicarboxylic acid failed to cause any significant decreases in the proportion of immature erythrocytes [P>0.01].

Mitomycin C did not cause any statistically significant decreases in the proportion [P>0.01] - it should be noted that even very cytotoxic compounds such as mitomycin C do not always produce a substantial decrease in this proportion as early as the 24 hour sampling time because of the lag caused by erythrocyte maturation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
In an in vivo mammalian erythrocyte micronucleus study conducted similar to OECD 474, according to GLP, 2,6-Naphthalenedicarboxylic acid (EC number 214-527-0 and CAS number 1141-38-4) did not cause any significant increase in the incidence of micronucleated immature erythrocytes or any substantial decrease in the proportion of immature erythrocytes in male and female mice, it is concluded that 2,6-Naphthalenedicarboxylic acid did not show any evidence of causing chromosome damage or bone marrow cell toxicity in this test system when administered by intraperitoneal injection.The maximum dose in the study (480 mg/kg by intraperitoneal injection) was considered to be a maximum tolerated dose as evidenced by mortality and clinical signs.