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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:
supporting study
Study period:
December 2006 - March 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Restrictions: 1. Dermal application, but no info on occlusion: thus oral exposure cannot be excluded, 2. No positive control is included, and 3. There is no mention of historical control data.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1. Dermal application, but no info on occlusion: thus oral exposure cannot be excluded, 2. No positive control is included, and 3. There is no mention of historical control data.
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
F344/NTac
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Male and female F344/NTac rats were obtained from the commercial colony at Taconic Farms, Inc. (Germantown, NY)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the rats were 5 to 6 weeks old
- Weight at study initiation: 82-84 g (female) 83-86 g (male)
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hardwood bedding, changed weekly, omnischield papaer cage filter changed every 2 weeks, Stainless steel Racks changed every 2 weeks.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 12 (males) or 13 (females) days

IN-LIFE DATES:
Date of First Dose: December 12 (males) or December 13 (females), 2006
Necropsy Date: March 13 (males) or March 14 (females), 2007

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72° ± 3° F (22° ± 1,668° C)
- Humidity (%): 50 ± 15
- Air changes (per hr): ≥10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
dermal
Vehicle:
- Vehicle(s)/solvent(s) used: ethanol
Details on exposure:
TEST SITE
- Area of exposure: dorsal surface just posterior to the scapulae to the base of the tail
- % coverage: Uncovered
- Type of wrap if used: Not specified
- Time intervals for shavings or clipplings: 1 week (shaved)

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg body weight (mice)
- Amount(s) applied (volume or weight with unit): 0.5 mL/kg body weight (rats)
- Concentration (if solution): 6.25%, 12.5%, 25%, 50%, or 100% (neat) OTNE
- Constant volume or concentration used: yes

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified
- Purity: USP-grade 95% ethanol

USE OF RESTRAINERS FOR PREVENTING INGESTION: Not used
Duration of treatment / exposure:
3 months
Frequency of treatment:
5 days per week
Post exposure period:
Sample collection time: 24 hours after the last dosing
Doses / concentrationsopen allclose all
Dose / conc.:
31.25 mg/kg bw/day
Remarks:
6.25% OTNE
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
12.5% OTNE
Dose / conc.:
125 mg/kg bw/day
Remarks:
25% OTNE
Dose / conc.:
250 mg/kg bw/day
Remarks:
50% OTNE
Dose / conc.:
500 mg/kg bw/day
Remarks:
100% OTNE
No. of animals per sex per dose:
5
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
Not included

Examinations

Tissues and cell types examined:
Peripheral blood erythrocytes from heparinized blood samples: micronucleated polychromatic erythrocytes (PCE) and mature erythrocytes (normochromatic erythrocytes (NCE). Approximately 1 x 10*6 erythrocytes (NCEs) and 20000 reticulocytes (PCEs) are scored for presence of micronuclei.

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The study was integrated into the 13 week subchronic study, so doses were chosen for the subchronic, not specifically for the micronucleus test

TREATMENT AND SAMPLING TIMES:
At the termination of the 3-month toxicity studies with OTNE, one to two drops of blood from male and female F344/NTac rats and male and female B6C3F1/N mice were collected in microtubes with EDTA and shipped on cool packs to the genetic toxicity testing laboratory for processing and fixation in ultracold methanol, as per procedures described in the MicroFlowBASIC Kit for rat blood samples or mouse blood samples (Litron Laboratories, Rochester, NY).

METHOD OF ANALYSIS:
FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) was used to carry out the analyses of the samples. Reticulocytes were identified by the presence of an active transferrin receptor (CD71+) on the cell surface and mature erythrocytes were CD71-negative. For the rat samples, only reticulocytes with the highest CD71 activity were evaluated due to the speed and efficiency with which the rat spleen removes damaged reticulocytes from circulation. Micronuclei were detected using propidium iodide (a DNA stain) in conjunction with RNase treatment.

Evaluation criteria:
In the micronucleus assay, a positive response is preferably based on the observation of both a significant trend as well as an observation of at least one dose group significantly elevated over the concurrent control group. If only one statistical test (trend or pairwise) is significant, the micronucleus assay is judged to be equivocal. The absence of both a significant trend and a significant dose results in a negative call for the assay. Ultimately, the scientific staff determines the final call after considering the results of statistical analyses, reproducibility of any effects observed, and the magnitudes of those effects.
Statistics:
Approximately 1 × 10^6 erythrocytes and 20,000 reticulocytes were scored per animal for presence of micronuclei. Based on prior experience with the large number of cells scored using flow cytometric scoring techniques (Kissling et al., 2007), it is assumed that the proportion of micronucleated reticulocytes is approximately normally distributed. The statistical tests selected for trend and for pairwise comparisons with the control group depend on whether the variances among the groups are equal. Levene’s test at α = 0.05 is used to test for equal variances. In the case of equal variances, linear regression is used to test for a linear trend with dose and Williams’ test is used to test for pairwise differences between each treatment group and the control group. In the case of unequal variances, Jonckheere’s test is used to test for linear trend and Dunn’s test is used for pairwise comparisons of each treatment group with the control group. To correct for multiple pairwise comparisons, the P value for each comparison with the control group is multiplied by the number of comparisons made. In the event that this product is greater than 1.00, it is replaced with 1.00. Trend tests and pairwise comparisons with the controls are considered statistically significant at P = 0.025.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Remarks:
The (vehicle) controls were used as negative controls
Negative controls validity:
valid
Positive controls validity:
not applicable
Remarks:
Information is derived from 90-day study
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No significant increases in micronucleated reticulocytes or erythrocytes were seen in male or female F344/NTac rats following exposure to OTNE, and no changes in the percentage of reticulocytes was observed in either sex of rat.

- Appropriateness of dose levels and route: The study was integrated into the 13 week subchronic dermal study, so doses and route were chosen for the subchronic, not specifically for the micronucleus test
Furthermore there were some methodological restrictions to the study; 1. Dermal application, but no info on occlusion: thus oral exposure cannot be excluded, making the exposure route and dose unclear, 2. No positive control is included, and 3. There is no mention of historical control data.

Any other information on results incl. tables

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Rats Following Dermal Application of OTNE for 3 Months

Male                
Dose (mg/kg bw/day) Number of Rats with Erythrocytes Scored Micronucleated PCEs/1,000 PCE P Value Micronucleated NCEs/1,000 NCE P Value PCEs (%) P Value
  0 5 0.38 ± 0.06 0.08 ± 0.01 1.20 ± 0.07  
OTNE 31.25 5 0.43 ± 0.08 0.431 0.07 ± 0.01 0.980 1.18 ± 0.03 1.000
  62.5 5 0.36 ± 0.03 0.509 0.04 ± 0.01 0.993 1.23 ± 0.03 1.000
  125 5 0.65 ± 0.07 0.021 0.05 ± 0.00 0.996 1.29 ± 0.07 1.000
  250 5 0.50 ± 0.06 0.022 0.05 ± 0.01 0.997 1.00 ± 0.08 0.022
  P = 0.045f P = 0.963f P = 0.025f  
Untreated control 5 0.38 ± 0.04 0.09 ± 0.01 1.12 ± 0.05  
OTNE 500 5 0.43 ± 0.09 0.320 0.04 ± 0.01 0.969 1.11 ± 0.07 0.850
Female                
Dose (%) Number of Rats with Erythrocytes Scored Micronucleated PCEs/1,000 PCE P Value Micronucleated NCEs/1,000 NCE P Value PCEs (%) P Value
  0 5 0.58 ± 0.15 0.08 ± 0.03 0.79 ± 0.07  
OTNE 31.25 5 0.40 ± 0.06 0.690 0.04 ± 0.01 1.000 0.84 ± 0.12 1.000
  62.5 5 0.55 ± 0.21 0.653 0.06 ± 0.02 1.000 1.07 ± 0.11 0.375
  125 5 0.58 ± 0.06 0.688 0.06 ± 0.01 1.000 0.69 ± 0.04 1.000
  250 5 0.52 ± 0.09 0.707 0.03 ± 0.00 1.000 0.70 ± 0.05 1.000
  P = 0.454f P = 0.910g P = 0.214g  
Untreated control 5 0.53 ± 0.07 0.07 ± 0.02 0.96 ± 0.06  
OTNE 500 5 0.45 ± 0.05 0.677 0.10 ± 0.02 0.116 1.29 ± 0.34 0.465

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, OTNE did not statistically significantly raise the frequency of micronucleated erythrocytes in male/female rats, and is not considered negative in this micronucleus test.
Executive summary:

OTNE was tested in the micronucleus test in rat to evaluate its genotoxic effect on peripheral blood erythrocytes. The test was performed as part of a repeated dose toxicity study, in a procedure comparable to OECD Guideline 474, and rated Klimisch 2 due to methodological restrictions to the study; 1. Dermal application, but no info on occlusion: thus oral exposure cannot be excluded, 2. No positive control is included, and  3. There is no mention of historical control data.

Five groups of rats, each comprising 5 males and 5 females, received dermal doses of 6.25, 12.5, 25, 50, and 100%. In rats these percentages resulted in estimated doses of 31.25, 62.5, 125, 250, and 500 mg/kg bw/day, 5 days per week for 3 months. Sample collection time started 24 hours after the last dosing. A vehicle (Ethanol) treated group, and an untreated group served as negative control. Observations were made of peripheral blood erythrocytes from heparinized blood samples. Furthermore, flow cytometric analysis was performed.  In the micronucleus assay, a positive response is preferably based on the observation of both a significant trend as well as an observation of at least one dose group significantly elevated over the concurrent control group. If only one statistical test (trend or pairwise) is significant, the micronucleus assay is judged to be equivocal. The absence of both a significant trend and a significant dose results in a negative call for the assay.

No significant increases in micronucleated reticulocytes or erythrocytes were seen in male or female F344/NTac rats following exposure to OTNE, and no changes in the percentage of reticulocytes was observed in either sex of rat. In the in 250 mg/kg bw/day male rats, the mean frequency of micronucleated reticulocytes was significantly (P < 0.025) elevated over the control in the two highest dose groups, but the magnitudes of the increases were very small, the trend test was not significant, and the response measured in the group that received the highest dose of OTNE (500 mg/kg bw/day) was lower than either of these two values; therefore, the response in male rat reticulocytes in the multidose test was judged to be negative. Under the conditions of this study, OTNE did not statistically significantly raise the frequency of micronucleated erythrocytes in male/female rats, and therefore is not considered to be negative in this micronucleus test.