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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
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to
Guideline:
other: MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3 (2000)
Deviations:
no
GLP compliance:
yes
Type of assay:
other: Mouse micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Purity: 97.04%
Impurities: Not reported

Test animals

Species:
mouse
Strain:
other: Crl:CD1(ICR)
Details on species / strain selection:
Mice have been shown to exhibit micronuclei indicative of broken chromosomes (clastogenic effects) or spindle effects (aneugenic effects) in response to known mutagens and were therefore used in this assay. The Crl:CD1(ICR) mouse was selected based on extensive experience with this strain at DuPont Haskell and its suitability for genetic toxicology studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories International, Inc. (Raleigh, North Carolina, U.S.A.)
- Age at study initiation: ~ 8 weeks old
- Weight at study initiation: Males: Solvent Control group: 31.2 g; Low-dose group: 31.1 g; Mid-dose group: 31.0 g; High-dose group: 31.6 g; and Positive control group: 31.0 g Females: Solvent Control group: 24.3 g; Low-dose group: 24.0 g; Mid-dose group: 24.2 g; High-dose group: 24.3 g; and Positive control group: 24.7 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Not mentioned
- Housing: All animals were housed in solid-bottom cages with Enrich-o’Cobs™ (i.e., enrichment-containing bedding).
- Diet (e.g. ad libitum): PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002
- Water (e.g. ad libitum): Tap water
- Acclimation period: At least 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26ºC (68-79ºF)
- Humidity (%): 30-70%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
0.1% Tween-80 in 0.5% aqueous methylcellulose
Duration of treatment / exposure:
All animals were given a single dose by oral gavage.
Frequency of treatment:
All animals were given a single dose by oral gavage.
Post exposure period:
No
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw (total dose)
Dose / conc.:
1 000 mg/kg bw (total dose)
Dose / conc.:
2 000 mg/kg bw (total dose)
No. of animals per sex per dose:
The vehicle control and the low- and mid-dose groups contained 10 animals/sex. The high-dose group contained 14 animals/sex.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CP)

Examinations

Tissues and cell types examined:
Bone marrow smears were prepared immediately after the sacrifice
Evaluation criteria:
Data were evaluated using scientific judgment taking into account both statistical and biological significance. Results not meeting the indicated criteria for positive or negative findings were evaluated on a case-by-case basis. Further investigation of an equivocal result was not required to obtain a conclusive finding.

The test substance was judged negative if the following conditions were met:

• No statistically significant dose-related increases in the group mean MN-RETs above the concurrent vehicle control value occurred at any concentration of the test substance.
• The MN-RET values of the test substance-treated animals were within reasonable limits of the laboratory historical control range.

The test substance was judged positive if the following conditions were met:

• The group mean MN-RETs was statistically significantly increased at one or more concentrations of the test substance compared to the concurrent vehicle control values.
• An accompanying statistically significant dose-response increase in MN-RETs was observed.

Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.
Statistics:
Micronucleus data was evaluated using scientific judgment taking into account both statistical and biological significance. The individual animal was considered the experimental unit. All micronucleus data analyses were one-tailed and conducted at a significance level of 5%. Data from the positive control group was not included in evaluating normality or variance homogeneity of distribution.

For any treatment groups where the increase in MN-RETs was found to be statistically significant, the data were further analyzed for dose response using the Cochran-Armitage trend test.

For each treatment group, the mean and standard deviation of % RETs and % MN-RETs were calculated. Data were be transformed prior to analysis using an arcsine square root or Freeman-Tukey function. This transformation is appropriate for proportions since the distribution of the transformed data more closely approximates a normal distribution than does the non-transformed proportion.

No statistical analysis was conducted on body weights or clinical signs.

See the section below for the Methods of Statistical Analyses used.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No clinical signs of toxicity were observed at any timepoint at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mortality occurred during the study.

No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint (See Tables 1 and 2 in the following section for details).

A statistically significant depression in the frequency of PCE/total erythrocytes was observed in female mice at the 48 hour timepoint indicating target cell exposure. No other reductions in PCE frequency were detected at any other timepoint in either male or female animals exposed to the test substance.

The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. There were no obvious changes in body weight or body weight gain in either male or female animals administered the test substance.

Any other information on results incl. tables

Table 1 - Micronucleus Evaluation for Male Mice

 

Parameter

Solvent Control

500 mg/kg Group

1000 mg/kg Group

2000 mg/kg Group

Positive Control Group

PCEs/1000 Erythrocytes

 

24-hour

 

48-hour

 

 

 

556 ± 14

 

565 ± 22

 

 

 

554 ± 43

 

a

 

 

 

563 ± 28

 

a

 

 

 

577 ± 16

 

562 ± 30

 

 

 

508* ± 17

 

b

PCE/NCE Ratio

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 1.255 ± 0.071

 

 

1.306 ± 0.112

 

 

 1.261 ± 0.229

 

 

a

 

 

 1.294 ± 0.149

 

 

a

 

 

 1.366 ± 0.092

 

 

1.289 ± 0.151

 

 

 1.032* ± 0.068

 

b

MNPCE/2000 PCEs

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 

3.0 ± 1.2

 

 

2.2 ± 1.9

 

 

 

2.0 ± 1.0

 

 

a

 

 

 

2.0 ± 1.0

 

 

a

 

 

 

1.8 ± 0.8

 

 

2.6 ± 1.9

 

 

 

35.3* ± 9.4

 

b

* Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test

a = Not evaluated at this timepoint

b = Group not included at this timepoint

 

Table 2 - Micronucleus Evaluation for Female Mice

 

PCEs/1000 Erythrocytes

 

24-hour

 

48-hour

 

 

 

566 ± 22

 

568 ± 11

 

 

 

568 ± 40

 

a

 

 

 

557 ± 16

 

a

 

 

 

557 ± 33

 

542* ± 12

 

 

 

516* ± 27

 

b

PCE/NCE Ratio

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 1.311 ± 0.127

 

 

1.317 ± 0.061

 

 

 1.333 ± 0.230

 

 

a

 

 

 1.258 ± 0.079

 

 

a

 

 

 1.265 ± 0.155

 

 

1.186* ± 0.059

 

 

 1.070* ± 0.118

 

b

MNPCE/2000 PCEs

 

24-Hour (Mean ± SD)

 

48-Hour (Mean ± SD)

 

 

 

2.8 ± 1.5

 

 

1.8 ± 1.5

 

 

 

1.6 ± 0.9

 

 

a

 

 

 

1.8 ± 1.3

 

 

a

 

 

 

3.2 ± 2.6

 

 

1.8 ± 0.8

 

 

 

26.6** ± 7.6

 

 

b

 * Statistically significant difference from control at p < 0.05 by Dunnett/Tamhane-Dunnett test

** Statistically significant difference from control at p < 0.05 by Dunn's test and the Dunnett/Tamhane-Dunnett test

a = Not evaluated at this timepoint

b = Group not included at this timepoint

Applicant's summary and conclusion

Conclusions:
All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in bone marrow polychromatic erythrocytes (PCEs) in male and female Crl:CD1(ICR) mice. Based on range-finding results, doses of 0, 500, 1000, and 2000 mg/kg of the test substance were selected for the main study. Concurrent control groups were administered 0.1% Tween-80 in 0.5% aqueous methylcellulose as the vehicle (negative) control, or 40 mg/kg of cyclophosphamide [positive control] (OECD Guidelines for the Testing of Chemicals No 474; EPA OPPTS Guideline 870.5395; EC Directive 2008/32/EC Method B.12; and MAFF Japan Notification No. 12-Nousan-8147 Guideline No. 2-1-19-3).

 

All animals were given a single dose by oral intubation. The vehicle control and the low- and intermediate-dose groups contained 10 animals/sex. The high-dose group contained 14 animals/sex. The positive indicator group consisted of 5 animals/sex. Half of the animals in each test substance and untreated control group were sacrificed at each timepoint, approximately 24 or 48 hours post-dosing, respectively. The positive control group was sacrificed approximately 24 hours post-dosing. Bone marrow smears were prepared immediately after the sacrifices. Two thousand PCEs per animal were evaluated for micronuclei and 1000 total erythrocytes per animal were evaluated for bone marrow toxicity.

 

Aliquots of the vehicle control and each test substance concentration were taken to confirm homogeneity, dose concentrations, and stability. Homogeneity and target concentrations were verified, and the test substance was stable for the duration of the dosing period.

 

No clinical signs of toxicity were observed at any timepoint at any dose level in male or female animals exposed to the test substance. No abnormalities were detected in the vehicle or positive control groups. No mortality occurred during the study.

 

No statistically significant increases in micronucleated PCE frequency were observed in any evaluated test substance-treated group of male or female animals at either timepoint (See Tables 1 and 2 in the "Other information on results including tables" section for details).

A statistically significant depression in the frequency of PCE/total erythrocytes was observed in female mice at the 48 hour timepoint indicating target cell exposure. No other reductions in PCE frequency were detected at any other timepoint in either male or female animals exposed to the test substance.

 

The positive control groups exhibited a response consistent with the micronucleated PCE historical control data. There were no obvious changes in body weight or body weight gain in either male or female animals administered the test substance.

 

All criteria for a valid study were met. Under the conditions of this study, the test substance did not induce biologically relevant increases in micronucleated polychromatic erythrocytes in animal bone marrow. The test substance was concluded to be negative in this in vivo study.