3,5-dimethylbenzoyl chloride

Genetic toxicity: in vivo

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:

6 June 1994 - 15 July 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP 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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

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:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

3,5-dimethylbenzoyl chloride

EC Number:

413-010-9

EC Name:

3,5-dimethylbenzoyl chloride

Cas Number:

6613-44-1

Molecular formula:

C9H9ClO

IUPAC Name:

3,5-dimethylbenzoyl chloride

Details on test material:

DMBC

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 Laboratories (Portage, MI.)
- Age at study initiation: Approximately 50 days old.
- Weight at study initiation: Approximately 21 - 28 g.
- Fasting period before study: 3 hours just prior to dose administration.
- Diet (e.g. ad libitum): ad libitum Purina Certified Rodent Chow 5002 C.
- Water (e.g. ad libitum): filtered tap water available ad libitum.
- Acclimation period: 13 days (including a 7 day quarantine period).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 24 °C.
- Humidity (%): 51 - 54 % relative.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil for DMBC.
Distilled water for the positive control.

Frequency of treatment:

A single oral dose administered by gavage.

Post exposure period:

Animals from test article and solvent control groups were killed at 24 or 48 hours after treatment. Animals from the positive control group were killed 24 hours after treatment.

Doses / concentrations

Remarks:

Doses / Concentrations:
1380, 690, 138 mg/kg at a volume of 10 mL/kg.
Basis:
actual ingested
Samples of the dosing solutions were submitted for independent chemical analysis of the test article concentration. The concentration did not deviate significantly from the expected target concentrations.

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control substance was Mitomycin-C (MMC) dissolved in distilled water at concentrations of 2.0 and 0.35 mg/kg; final concentration, 10 mL/kg .
The positive control was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.

Examinations

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

The doses for this study were selected after evaluating the results from an acute oral toxicity study in male and female CD-1 mice. In the acute study, LD50 estimation was 2475 mg/kg and the LD10 estimation for male and female mice was 1380 mg/kg. Therefore, the maximum concentration for this study was 1380 mg/kg as this dose was expected to produce significant toxicity without leading to lethality. In addition to the maximum dose level, 690 mg/kg and 138 mg/kg of DMBC were used.

Animals were killed by cervical dislocation at approximately 24 and 48 hours after dosing. In the high dose group, 2 additional animals per time point were dosed to account for the possibility of unexpected deaths. The positive control groups were killed 24 hours after dosing. Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice.

Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70 % ethanol. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15 mL centrifuge tube, containing approximately 1 mL of Foetal Bovine Serum (FBS) using a 1 cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x gravity for 5 minutes, and the supernatant was removed, leaving approximately 0.1 mL above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed. A small drop of the cell suspension (approximately 10 µL) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridine Orange staining solution.

Scoring
Slides from at least five animals per dose group were observed. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain.
Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. For each animal, a total of at least 1000 polychromatic erythrocytes and normochromatic erythrocytes were recorded to calculate the PCE/NCE ratio. For each animal a total of at least 2000 polychromatic erythrocytes were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.

Cell counting was accomplished using the Xybion Path/Tox computer software system, G Module (GICELL program) which captures data from the cell counter keyboard and provides an audit trail for quality assurance.

Evaluation criteria:

An assay is considered acceptable for evaluation if the following criteria are met:
-The highest dose of the positive control elicits a significant increase in the average number of MN-PCE per 1000 PCE when compared to the negative controls.
-At least three animals from each sex must be alive at the time of sacrifice for each dose level.

Data was analysed separately for male and female animals. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analysed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of:
-An overall effect of DMBC treatment relative to control
-A linear dose-response trend among the DMBC groups
-A quadratic dose-response trend among the DMBC groups. Additionally, pairwise comparisons between each of the three DMBC groups and the control group were made using Dunnett's t-test (Kirkland, D.J., 1989).

Statistics:

The test article is considered positive in this assay if it elicits a positive dose-response and a statistically significant increase over that of the concurrent vehicle control in the number of MN-PCE at one or more dose levels. In the event that the test article elicits a statistically significant increase in the number of MN-PCE due to an unusually low number of MN-PCE in the concurrent vehicle control, the data from that dose may be compared to historical vehicle control data. In the event there is no positive dose-response, at least two consecutive treatment groups should elicit a statistically significant increase in the number of MN-PCE when compared to the vehicle control.

A test article is considered negative in this assay if no indication of a positive dose-response is observed and the treatment groups do not show a statistically significant increase in the number of MN-PCE when compared to the vehicle control.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Clinical Signs
Table 1 summarises the toxicologic signs observed in the animals. Clinical signs were consistent with signs observed in an acute oral toxicity study and indicated a significant dose-related increase in toxicity at the high dose in both males and females. Several animals treated with 1380 mg/kg DMBC were observed to be passive after dose administration on day 0. Tremors and prostrate body position were observed in one female from the same dose group. Passiveness, ataxia, laboured breathing and ptosis were observed in the same animal on day 1. No toxicologic signs were observed in any of the remaining animals on day 2. No toxicologic signs were observed in any of the other dose groups.

Micronucleus Evaluation
The results are summarised in Table 2. The test article did not induce a significant increase in the frequency of polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the solvent control (this was true for both the 24 and 48 hour time points). There was no statistically significant change in the polychromatic/ normochromatic ratio.

A significant increase in the frequency of polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, MMC, when compared to the solvent control. In addition, a significant increase was observed in the bone marrow cells of mice treated with 0.35 mg/kg MMC, indicating the sensitivity of the assay to detect induced genetic damage.

Any other information on results incl. tables

Table 1 Summary of Clinical Signs

Dose Group	No. of Animals Exhibiting signs
	Day 0	Day1	Day2
10 mL/kg Corn oil   Male Female	0/10 0/10	0/10 0/10	0/5 0/5
1380 mg/kg DMBC   Male            Passive   Female       Passive Tremors Prostrate Ataxic Ptosis Laboured breathing	5/14     1/14 1/14 1/14 0/14 0/14 0/14	0/14     1/14 0/14 0/14 1/14 1/14 1/14	0/7     0/7 0/7 0/7 0/7 0/7 0/7
690 mg/kg DMBC   Male Female	0/10 0/10	0/10 0/10	0/5 0/5
138 mg/kg DMBC   Male Female	0/10 0/10	0/10 0/10	0/5 0/5
0.35 mg/kg MMC   Male Female	0/5 0/5	0/5 0/5	- -
2.0 mg/kg MMC   Male Female	0/5 0/5	0/5 0/5	- -

Day 0 = Treatment day

Fractions represent number of animals exhibiting the signs / total number of animals in the group.

 

Table 2 Mean Summary Data for Male Animals

Dose (mg/kg)	Day		PCE Total	MN-PCE	MN-PCE %	PCE/NCE Ratio
Control	1       2	Mean SD N   Mean SD N	2105 45 5   2077 78 5	3 1 5   2 1 5	0.14 0.05 5   0.08 0.06 5	1.35 0.66 5   1.51 0.46 5
138	1       2	Mean SD N   Mean SD N	2069 52 5   2107 36 5	2 1 5   1 0 5	0.10 0.06 5   0.04 0.02 5	1.40 0.82 5   1.64 0.54 5
690	1       2	Mean SD N   Mean SD N	2079 38 5   2072 32 5	3 1 5   2 2 5	0.13 0.06 5   0.11 0.08 5	1.43 0.24 5   1.46 0.74 5
1380	1       2	Mean SD N   Mean SD N	2080 63 7   2085 38 7	3 2 7   3 2 7	0.12 0.09 7   0.12 0.08 7	1.67 0.61 7   1.63 0.69 7
MMC 0.35	-	Mean SD N	2047 22 5	13 4 5	0.62* 0.18 5	1.78 0.54 5
MMC 2	-	Mean SD N	2063 29 5	137 21 5	6.65* 1.07 5	1.25 0.65 5

SD = Standard Deviation                   

N = Number of Observations

NCE = Normochromatic Erythrocytes                              

MN-PCE = Micronucleated Polychromatic Erythrocytes             

PCE = Polychromatic Erythrocytes

*Indicates a Statistically Significant Difference from Control (p<0.05).

 

Mean Summary Data for Female Animals

Dose (mg/kg)	Day		PCE Total	MN-PCE	MN-PCE %	PCE/NCE Ratio
Control	1       2	Mean SD N   Mean SD N	2086 47 5   2077 42 5	5 3 5   2 2 5	0.22 0.17 5   0.10 0.09 5	1.52 0.43 5   1.56 0.73 5
138	1       2	Mean SD N   Mean SD N	2089 62 5   2083 51 5	2 2 5   2 2 5	0.08 0.10 5   0.12 0.10 5	1.53 0.62 5   1.85 0.70 5
690	1       2	Mean SD N   Mean SD N	2073 31 5   2114 53 5	2 1 5   1 1 5	0.12 0.04 5   0.07 0.05 5	1.32 0.68 5   1.90 0.75 5
1380	1       2	Mean SD N   Mean SD N	2100 92 7   2095 74 7	4 2 7   2 2 7	0.20 0.11 7   0.09 0.09 7	1.19 0.61 7   1.54 0.63 7
MMC 0.35	-	Mean SD N	2018 37 5	12 5 5	0.55* 0.21 5	1.67 0.66 5
MMC 2	-	Mean SD N	2043 25 5	135 25 5	6.64* 1.28 5	1.21 0.07 5

SD = Standard Deviation                    

N = Number of Observations

NCE = Normochromatic Erythrocytes                              

MN-PCE = Micronucleated Polychromatic Erythrocytes             

PCE = Polychromatic Erythrocytes

*Indicates a Statistically Significant Difference from Control (p<0.05).

 

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Under the conditions of this study, DMBC is not mutagenic in the Micronucleus Assay in CD-1 mouse bone marrow cells.

Executive summary:

DMBC was evaluated for potential to induce genetic damage in vivo, using the micronucleus assay in mouse bone marrow cells in accordance with OECD Guideline 474, U.S. EPA 40 CFR Part 158.340 and Guideline 84-2.

  

Adult CD-1 male and female mice received a single oral dose of the test article in corn oil at concentrations of 1380, 690 or 138 mg/kg. Control animals received a single oral dose of corn oil (solvent control), or an intraperitoneal injection of 0.35 or 2.0 mg/kg mitomycin-C (positive control). Animals from test article and solvent control groups were killed at 24 or 48 hours after treatment. Animals from the positive control group were killed 24 hours after treatment. Bone marrow slides were prepared and the frequency of micronucleated polychromatic erythrocytes (MN-PCE) was measured as an indicator of cytogenetic damage. In addition, the polychromatic/normochromatic (PCE/NCE) ratio was measured to evaluate the cytotoxicity of the test agent.

 

Both male and female mice treated with DMBC exhibited clinical signs of systemic toxicity up to 24 h after treatment. By 48 h after treatment the animals had recovered and no clinical signs were observed.

 

The test article did not induce a significant increase in the frequency of polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the solvent controls. There was no statistically significant change in the PCE/NCE ratio.

 

Under the conditions of this study, DMBC is not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.