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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: oral exposure cannot be excluded. 2) Dose levels are high; top dose twice the recommended dose, all doses used induced (local) toxicity, higher doses induced systemic toxicity. 3) No positive control is included 4) No mention of historical control data 5) No bone marrow was collected.

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:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
1) Dermal application, but no info on occlusion. 2) Dose levels are high; top dose twice the recommended dose 3) No positive control is included 4) No mention of historical control data
GLP compliance:
yes
Type of assay:
other: in vivo micronucleus assay

Test material

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

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Male and female B6C3F1/N mice were obtained from the NTP colony at Taconic Farms, Inc
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: Upon start of the study the mice were 6 to 7 weeks old
- Weight at study initiation: Male 22.6-23.7 g, Female 18.4-19.1 g
- Housing: 1 animal/cage, polycarbonate cage, bedded with irradiated heat-treated Sani-Chip hard wood 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, Irradiated NTP-2000 pelleted diet (Zeigler Brothers, Inc., Gardners, PA), available ad libitum, changed weekly
- Water (e.g. ad libitum): ad libitum, Tap water (Washington Suburban Sanitary Commission, Potomac Plant) via automatic watering system (Edstrom Industries, Inc., Waterford, WI)
- Acclimation period: 14 (males) or 15 (females) days

IN-LIFE DATES (MICE)
Date of First Dose: December 14 (males) or December 15 (females), 2006
Necropsy Date: March 15 (males) or March 16 (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)
- 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.:
125 mg/kg bw/day
Remarks:
6.25% OTNE
Dose / conc.:
250 mg/kg bw/day
Remarks:
12.5% OTNE
Dose / conc.:
500 mg/kg bw/day
Remarks:
25% OTNE
Dose / conc.:
1 000 mg/kg bw/day
Remarks:
50% OTNE
Dose / conc.:
2 000 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) & 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 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 (CD7 1+) on the cell surface and mature erythrocytes were CD71-negative. For the rat samples, only retic ulocytes 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 cons idering 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 mouse 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 resultsopen allclose all
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
Systemic Liver effects observed for the all doses. Haematology effects at 2000 mg/kg bw. Skin effects observed for all dose groups.
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
Key result
Sex:
female
Genotoxicity:
other: inconclusive
Toxicity:
yes
Remarks:
Systemic Liver effects observed at all doses. Haematology effects at 2000 mg/kg bw. Skin effects observed for all dose groups.
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
Remarks on result:
other: The increase at 2000 mg/kg bw is not considered relevant as this is twice the maximum dose. At the next lower dose the slight positive results cannot be interpreted in absence of (historical) control values.
Additional information on results:
RESULTS OF DEFINITIVE STUDY

- Induction of micronuclei (for Micronucleus assay):
Male mice: Minimal increase in NCE micronuclei for 100% OTNE, no dose response relationship. Although this increase was statistically significant (P = 0.001), the magnitude of the increase was minimal (1.45 ± 0.02 in the control versus 1.59 ± 0.03 in the treated group), and there was no dose response effect. Therefore, the result was judged to be equivocal. No other significant effects were noted.
Female mice: Single dose increase in PCE micronuclei (P=0.005, 100% OTNE), dose dependent increase in NCE micronuclei (P<0.001, 0-50% OTNE), as well as single dose increase (P<0.001, 100% OTNE).

In the original study report, the results of the micronucleus assay were considered equivocal in male mice and positive in female mice after exposure to OTNE. However, the top dose in mice (2000 mg/kg bw/day) that describes the effect, is twofold the recommended dose of the OECD Test Guideline 474. Furthermore, the test results for the high doses are accompanied with the observation of systemic and local toxicity. In light of these restictions, the assessors consider it necessary to evaluate the effects by evaluating the 125, 250, 500, 1000 mg/kg bw/day groups only. In that case, only a very slight increase in micronucleated mature erythrocytes is observed in female rats at 1000 mg/kg bw/day. This very small increase (compared to the vehicle controls), calls into question the biological significance of the observation. As no historical control reference data is available, OTNE was concluded to be negative in male mice and equivocal in female mice for cytogenicity.

- Ratio of PCE/NCE (for Micronucleus assay):
Male mice: The %PCEs was not statistically different from the control group.
Female mice: The %PCEs was significantly increased in female mice exposed to 100% OTNE (1.08 ± 0.15 in the control versus 1.89 ± 0.25 in the treated group) however, the absolute %PCEs in the 100% OTNE group was equal to or less than those seen in the 12.5% to 50% OTNE-treated female mice, and no dose-dependent indication was observed. Considering all the data, the increase in the percentage of reticulocytes in the 100% OTNE group was not considered to be biologically significant, thus not indicating of stimulation of erythropoiesis, or bonemarrow toxicity.

- 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

Any other information on results incl. tables

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

Male                
  Dose (%) Number of Rats with Erythrocytes Scored Micronucleated PCEs/1,000 PCE P Value Micronucleated NCEs/1,000 NCE P Value PCEs (%) P Value
Vehicle control 0 5 2.42 ± 0.16 1.47 ± 0.02 1.46 ± 0.08  
OTNE 6.25 5 2.55 ± 0.24 0.397 1.48 ± 0.04 0.703 1.52 ± 0.12 0.620
  12.5 5 2.78 ± 0.30 0.471 1.43 ± 0.04 0.787 1.58 ± 0.08 0.651
  25 5 2.43 ± 0.20 0.501 1.39 ± 0.05 0.820 1.52 ± 0.04 0.695
  50 5 2.24 ± 0.14 0.518 1.49 ± 0.02 0.477 1.47 ± 0.05 0.717
 Trend significance P = 0.865e P = 0.442 P = 0.851  
Untreated control 5 2.66 ± 0.14 1.45 ± 0.02 1.60 ± 0.06  
OTNE 100 5 2.29 ± 0.15 0.805 1.59 ± 0.03 0.001 2.00 ± 0.20 0.066
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
Vehicle control 0 5 2.13 ± 0.12 1.01 ± 0.02 1.52 ± 0.12  
OTNE 6.25 5 1.76 ± 0.10 0.877 1.01 ± 0.02 0.472 1.51 ± 0.09 1.000
  12.5 5 2.15 ± 0.05 0.887 1.07 ± 0.04 0.064 2.06 ± 0.13 0.053
  25 5 1.95 ± 0.14 0.912 1.08 ± 0.02 0.039 1.84 ± 0.21 0.056
  50 5 1.85 ± 0.12 0.924 1.15 ± 0.01 0.001 1.96 ± 0.14 0.045

 Trend

significance

P = 0.856 P < 0.001 P = 0.050  
Untreated control 5 1.98 ± 0.14 1.03 ± 0.04 1.08 ± 0.15  
OTNE 100 5 2.88 ± 0.23 0.005 1.27 ± 0.01 < 0.001 1.89 ± 0.25 0.031

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, OTNE was concluded to be negative in male mice and inconclusive in female mice for cytogenicity.
Executive summary:

OTNE was tested in the micronucleus test in mice 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 significant oral exposure occurred; 2) Dose levels are high; top dose twice the recommended dose, all doses used induced (local) toxicity, highest doses induced systemic toxicity; 3) No positive control is included due to the results retrieved from a 90 -day study and; 4) There is no mention of historical control data.

Five groups of mice, each comprising 5 males and 5 females, received dermal doses of 6.25, 12.5, 25, 50, and 100% OTNE. In mice these dosing percentages resulted in estimated doses of 125, 250, 500, 1000 and 2000 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.

A statistically significant slight increase in the frequency of micronucleated mature erythrocytes was observed only in male mice exposed to OTNE at 2000 mg/kg bw/day (1.45 ± 0.02 in the control versus 1.59 ± 0.03 in the treated group). In female mice, OTNE did not increase the frequency of micronucleated reticulocytes in the 125, 250, 500 and 1000 mg/kg bw/day groups. Micronucleated mature erythrocytes showed a very slight but significant increase in the 1000 mg/kg bw/day group (1.01 ± 0.02 in the control versus 1.15 ± 0.01  in the treated group), this increase was observed to be dose-dependent (P<0.001). In the 2000 mg/kg bw/day group, micronucleated reticulocytes were significantly increased (P = 0.005) in female mice. An increase in micronucleated mature erythrocytes was also observed in the 2000 mg/kg bw/day group (1.03 ± 0.04 in the control versus 1.27 ± 0.01 in the treated group).

In the original study report, the results of the micronucleus assay were considered equivocal in male mice and positive in female mice after exposure to OTNE. However, the top dose in mice (2000 mg/kg bw/day) that describes the effect, is twofold the recommended dose of OECD Test Guideline 474. Furthermore, the test results for the high doses are accompanied with the observation of systemic and local toxicity in red blood cell parameters. In light of these limitations, the assessors consider only the effects at the 125, 250, 500 and 1000 mg/kg bw/day groups as toxicologically relevant. In that case, only a very slight increase in micronucleated mature erythrocytes is observed in female rats at 1000 mg/kg bw/day. This very small increase (compared to the vehicle controls), calls into question the biological significance of the observation. No historical control reference data is available, which makes it impossible to evaluate the relevance of this effect. Based on the previous, OTNE was concluded to be negative in male mice and inconclusive in female mice in view of absence of (historical) control values.