Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro tests

- Gene mutation in bacteria (OECD TG 471): negative

- In vitro cytogenicity (OECD TG 473): negative

- In vitro gene mutation (OECD TG 476): negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 November 1994- 21 February 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not relevant
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 tissue culture medium
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
First test:
Without S-9 mix: 3.75, 7.5, 15, 20, 30, 40, 50, 60, 80 and 100 ug/ml
With S-9 mix: 7.8, 15.6, 31.25, 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Second test:
Without S-9 mix, 18 hour harvest: 3.75, 7.5, 15, 20, 30, 40, 50, 60, 80 and 100 ug/ml
With S-9 mix, 18 hour harvest: 15.6, 31.25, 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Without S-9 mix, 32 hour harvest: 7.5, 15, 20, 30, 40, 50, 60 and 80 ug/ml
With S-9 mix, 32 hour harvest: 62.5, 75, 100, 125, 150, 200 and 250 ug/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulphonate (direct-acting mutagen) and Cyclophosphamide (promutagen)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Test 1: with S-9: 3 hours; without S-9: continuous exposure: 18 hours
Test 2: with S-9: 3 hours; without S-9: 18 or 32 hours
- Fixation time (start of exposure up to fixation or harvest of cells):
Test 1: 18 hours
Test 2: 18 or 32 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine, 0.25 μg/ml., last 2 hours of incubation
STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: duplicate cultures for each treatment

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads from each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: the dose level causing a decrease in mitotic index of approximately 50% of the solvent control value was used as the highest dose level for the metaphase analysis.

OTHER: Prior to commencing testing, the solubility of the test substance was assessed. OTNE was found to be miscible with DMSO at 125 ug/ml. When added to culture medium at 1% v/v, to give a final concentration of 1250 ug/ml, small oily globules were formed. The highest concentration chosen for subsequent testing, 1000 ug/ml, was at the limit of solubility since only a slight amount of insoluble test substance was present.
Evaluation criteria:
No data
Statistics:
Fisher's test. (FISHER, R.A. (1973) The Exact Treatment of 2 x 2 Table in: Statistical Methods for Research Workers. Hafner Publishing Company, New York).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Test 1:
In the absence of S-9 mix, OTNE caused a decrease in the mitotic index (MI) to 27% of the solvent control value at 62.5 ug/ml. However, there were insufficient metaphase figures available for analysis. As no other concentration caused a suitable reduction in the MI, the test was repeated using a narrower range of dose levels. In the repeat test, 50 ug/ml caused a reduction in the MI to 55% of the solvent control value. This concentration, together with 30 and 15 ug/ml, was selected for further analysis.
In the presence of S-9 mix, OTNE caused a decrease in MI to 67% of the solvent control value at 125 ug/ml. However, there were insufficient metaphase figures suitable for analysis. As no other concentration caused a suitable reduction in the MI, the test was repeated with a narrower range of dose levels. In the repeat test 125 ug/ml caused a reduction in the MI to 40% of the solvent control value. This concentration, together with 62.5 and 15.6 ug/ml was selected for further analysis.
Test 2: 18 hour harvest
In the absence of S-9 mix, OTNE caused a dose-related decrease in MI. The highest concentration with sufficient metaphase figures suitable for analysis, 30 ug/ml, caused a reduction in the MI to 58 % of the solvent control value. Therefore, the dose levels chosen for metaphase analysis were 30, 15 and 7.5 ug/ml.
In the presence of S-9 mix, OTNE, at a concentration of 125 ug/ml, caused a reduction in the MI to 61 % of the solvent control value. All higher dose levels were too toxic for analysis. Therefore, the dose levels chosen for metaphase analysis were 125, 62.5 and 31.25 ug/ml.
Test 2 - 32 hour harvest
In the absence of S-9 mix, although the MI was reduced to 49% of control levels at 30 ug/ml, there were insufficient metaphase figures suitable for analysis. The highest analysable concentration, 15 ug/ml, reduced the MI to 73% of the solvent control value. Therefore, 15 ug/ml was chosen for metaphase analysis.

COMPARISON WITH HISTORICAL CONTROL DATA: Any increase in the number of aberrant cells lies within the historical control range.
Remarks on result:
other: all strains/cell types tested

Except for one test, there were no statistically significant increases in the proportion of metaphase figures with chromosomal aberrations, at any concentration analysed, in the presence and absence of S-9 mix. In the second test -18 hour harvest - in the absence of S-9 mix, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase, to 4.0%, lies within the historical control range of 0-5.25 %, there was no dose-response relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related.

Conclusions:
There were no statistically significant increases, treatment-related, in the proportion of aberrant cells, in the presence and absence of S-9 mix, at the 18 or 32 hour harvests. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
Executive summary:

A study was performed to assess the ability of OTNE to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time, cell division was arrested using Colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of OTNE to cultured human lymphocytes, the mitotic index (MI) was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis:

First test:

Without S-9 mix, 18 hour harvest: 15, 30 and 50 ug/ml

With S-9 mix, 18 hour harvest: 15.6, 62.5 and 125 ug/ml

Second test:

Without S-9 mix, 18 hour harvest: 7.5, 15 and 30 ug/ml

With S-9 mix, 18 hour harvest: 31.25, 62.5 and 125 ug/ml

Without S-9 mix, 32 hour harvest: 15 ug/ml

With S-9 mix, 32 hour harvest: 75 ug/ml

There were no statistically significant increases in the proportion of aberrant cells, in the presence of S-9 mix, at the 18 or 32 hour harvests. In the absence of S-9 mix, in the second test at the 18 hour harvest, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase lies within the historical control range, there was no dose-relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related.

All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells.

It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 July - 2 September 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
other: ICH (1996) Guideline S2A: : Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals.
Qualifier:
according to
Guideline:
other: ICH (1998) Guideline S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals.
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
thymidine kinase locus, TK +/-.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media:

R0 RPMI 1640, buffered with 2 mg/mL sodium bicarbonate, supplemented with 2.0 mM L-glutamine and 50 µg/mL gentamicin.
R10p R0, supplemented with 0.1% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 10% v/v.
R30p R0, supplemented with 0.02% v/v Synperonic F68, 1.0 mM sodium pyruvate and HiDHS at 30% v/v.

R10p medium was used for cell culture unless otherwise specified.

R20p medium was used for the cloning efficiency plating. This was prepared by mixing equal volumes of R10p and R30p.

Selective medium consisted of R10p containing 4 µg/mL trifluorothymidine (TFT).

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
other: heterozygous at the thymidine kinase locus, TK +/-.
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Preliminary toxicity test: 4.57, 9.15, 18.3, 36.59, 73.19, 146.38, 292.75, 585.5, 1171 and 2342 µg/mL

Mutation tests: -S9 mix Test 1 (3 hours) 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL
+S9 mix Test 1 (3 hours) 10, 30, 50, 70, 90, 110, 130, 150 and 200 µg/mL
-S9 mix Test 2 (24 hours) 10, 20, 30, 40, 50, 60, 70 and 80 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol

- Justification for choice of solvent/vehicle:

test substance soluble at 234.12 mg/ml in ethanol
a solution of 2342 mg/mL, dosed at 1% in medium, showed no precipitate or change in osmality in the culture medium.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
10 µg/ml main test 1, 3 hr treatment in absence of S9 mix. 5 µg/ml main test 2, 24 hr treatment, in the absence of S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
1 µg/ml main test 1, 3hr treatment, in presence of S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 hr and 24 hr
- Expression time (cells in growth medium): 24 and 48 hr
- Fixation time (start of exposure up to fixation or harvest of cells): 7 days

NUMBER OF REPLICATIONS:
Preliminary test - 2 (vehicle control), 1 (test levels)
Main test 1 and 2 - 4 (vehicle control), 2 (test), 2 (positive control)

NUMBER OF CELLS EVALUATED:
Preliminary test - 6x10^6
Main test 1 (3hr exposure)- 1.2x10^7
Maintest 1 (24 hr exposure) - 3x10^6

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Evaluation criteria:
On completion of each main mutagenicity test, data were examined for cell growth parameters, cytotoxicity, plating efficiencies, spontaneous and positive control MF, and percent small colonies in positive control cultures.
Statistics:
The data were analysed using Fluctuation application SAFEStat (SAS statistical applications for end users) version 1.1.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: was observed by eye at the end of treatment in the preliminatry test at concentrations of 146.38 µg/mL and greater and 292.75 µg/mL and greater in the absence and presence of S9 mix, respectively, following a 3 hour exposure.


RANGE-FINDING/SCREENING STUDIES: Toxicity was observed in the preliminary toxicity test. Following a 3 hour exposure to OTNE at concentrations from 4.57 to 2342 µg/mL, relative suspension growth (RSG) was reduced from 97 to 0% and from 124 to 0% in the absence and presence of S9 mix respectively. Following a 24 hour exposure in the absence of S9 mix RSG was reduced from 111 to 0%. The concentrations assessed for determination of mutant frequency in the main test were based upon these data, the objective being to assess concentrations which span the complete toxicity range of approximately 10 to 100% relative total growth (RTG).


COMPARISON WITH HISTORICAL CONTROL DATA:

Historical control data are provided for 3 hr exposure in the presence and absence of S9 mix and for 24 hr exposure in the absence of S9 mix.







Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 40 and 90 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 20 and 23% respectively.

In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 50 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed and RTG was reduced to 16%.

Conclusions:
It was concluded that OTNE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.
Executive summary:

In an in vitro mammalian cell gene mutation test which was performed in accordance with OECD476 and under GLP conditions, the mutagenic properties of OTNE were determined in mouse lymphoma cells. Based on the preliminary toxicity test, the mutation tests were performed using the following concentrations: Test 1: without S9 mix (3 hours) 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL, with S9 mix (3 hours) 10, 30, 50, 70, 90, 110, 130, 150 and 200 µg/mL, Test 2: without S9 mix (24 hours) 10, 20, 30, 40, 50, 60, 70 and 80 µg/mL. Exposure to solvent ethanol was included as a negative control, and methylmethanesulfate and benzo(a)pyrene were included as positive control substances.

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 40 and 90 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 20 and 23% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 50 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed and RTG was reduced to 16%.

It was concluded that OTNE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 November 1994 - 29 November 1994
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
bacterial reverse mutation assay
Target gene:
His-gene: Amino acid histidine, and Trp-gene: amino acid tryptophan
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 , TA 100 and E. coli WP2 uvrA
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: S. typhimurium TA1535, TA1537, TA1538: rfa, uvrB; S. typhimurium TA98, TA100: rfa, uvrB, pKM101; E. coli WP2: uvrA
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 induced by Aroclor 1254
Test concentrations with justification for top dose:
5000, 2500, 1250, 625, and 312.5 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance was virtually insoluble in water.
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9: S. typhimurium TA 1535, TA 100, E. coli WP2 uvrA: N-Ethyl-N'-nitro-N-nitrosoguanidine; S. typhimurium TA 1537: 9-Aminoacridine; S. typhimurium TA 1538, TA 98: 2-Nitrofluorene. +S9: all strains: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Selection time (if incubation with a selection agent): 3 days

SELECTION AGENT (mutation assays): agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: 3 plates per concentration

NUMBER OF CELLS EVALUATED: 10*9 viable cells

DETERMINATION OF CYTOTOXICITY
- Method: determination of a concentration related reduction in the mean number of revertants per plate and/or the reduction of the microcolony background lawns

OTHER EXAMINATIONS: Preliminary toxicity/Dose range finding test at 5, 50, 500, and 5000 ug/plate.
Evaluation criteria:
If treatment with a test substance produces an increase in revertant colony numbers of at least twice the concurrent solvent controls, with some evidence of a positive dose-relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S-9 mix, it is considered to show evidence of mutagenic activity in this test system. No statistical analysis is performed.
If treatment with a test substance does not produce reproducible increases of at least 1.5 times the concurrent solvent controls, at any dose level with any bacterial strain, it is considered to show no evidence of mutagenic activity in this test system. No statistical analysis is performed.
If the results obtained fail to satisfy the criteria for a clear "positive" or "negative" response, the following approach is taken in order to resolve the issue of the substance's mutagenic activity in this test system.
- Repeat tests may be performed using modifications of the experimental method.
- If no clear "positive" response can be obtained the test data may be subjected to analysis to determine the statistical significance of any observed increases in revertant colony numbers.
Statistics:
Mean and Standard Deviation
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 , TA 100 and E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: OTNE was not toxic towards the tester strains in the preliminary toxicity test. Therefore 5000 ug/plate was chosen as the top dose level in the mutation tests.
Remarks on result:
other: all strains/cell types tested
Conclusions:
No substantial increases in revertant colony numbers of any of the tester strains were observed following treatment with OTNE at any dose level, in the presence or absence of S-9 mix, in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO shows no evidence of mutagenic activity in this bacterial system.
Executive summary:

In this in vitro assessment of the mutagenic potential of OTNE, histidine dependent auxotrophic mutants of S. typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of E. Coli (WP2uvrA) were exposed to the test substance, diluted in DMSO which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 ug/plate, no toxicity was observed. A top dose level of 5000 ug/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, and 312.5 ug/plate. No evidence of mutagenic activity was seen at any dose level of OTNE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO was not mutagenic in this bacterial system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus similar to OECD TG 474 in rat (as part of the dermal 90 -day NTP study): Negative in male/female mice

In vivo micronucleus similar to OECD TG 474 in mice (as part of the dermal 90 -day NTP study): Negative in male mice; inconclusive in female mice due to absence of proper controls.

Link to relevant study records

Referenceopen allclose all

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.
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
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
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
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
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.
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

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
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.

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.
Qualifier:
equivalent or similar to
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
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
F344/NTac
Sex:
male/female
Details on test animals 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
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
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
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.
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.

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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

The three available in vitro studies in the dossier were used as key studies. The Ames, chromosome aberration (CA) and mouse lymphoma test all indicate that OTNE is not genotoxic. An in vivo micronucleus test performed as part of a 90-day dermal repeated dose toxicity study in rats and mice is also available. The test is enclosed as a Klimisch 2 study, but is only considered to be supporting value to the in vitro results, due to the following methodological restrictions to the study: 1) Dermal application, but no info on occlusion, thus oral exposure cannot be excluded; 2) Dose levels in mice are high: the top dose is twice the recommended dose, all doses used induced (local) toxicity, highest doses induced systemic toxicity; 3) No positive control is included and;  4) There is no mention of historical control data. Based on the available dataset as detailed below, OTNE is considered to be non-genotoxic.

Gene mutation in bacteria

In the in vitro assessment of the mutagenic potential of OTNE in bacteria, histidine dependent auxotrophic mutants of S. typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) and a tryptophan dependent mutant of E. Coli (WP2uvrA) were exposed to the test substance, diluted in DMSO which was also used as a negative control. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats. In the preliminary dose range finding study with dose levels of up to 5000 ug/plate, no toxicity was observed. A top dose level of 5000 ug/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 2500, 1250, 625, and 312.5 ug/plate. No evidence of mutagenic activity was seen at any dose level of OTNE in either mutation test. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It is concluded that OTNE tested in DMSO was not mutagenic in this bacterial system.

In vitro cytogenicity assay (chromosomal aberrations)

Another in vitro study was performed to assess the ability of OTNE to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time, cell division was arrested using Colchicine, the cells harvested and slides prepared, so that metaphase figures could be examined for chromosomal damage.

In order to assess the toxicity of OTNE to cultured human lymphocytes, the mitotic index (MI) was calculated for all cultures treated with the test substance and the solvent control. On the basis of these data, the following concentrations were selected for metaphase analysis. First test: Without S-9 mix, 18 hour harvest: 15, 30 and 50 ug/ml; with S-9 mix, 18 hour harvest: 15.6, 62.5 and 125 ug/ml. Second test: Without S-9 mix, 18 hour harvest: 7.5, 15 and 30 ug/ml; with S-9 mix, 18 hour harvest: 31.25, 62.5 and 125 ug/ml; without S-9 mix, 32 hour harvest: 15 ug/ml; with S-9 mix, 32 hour harvest: 75 ug/ml.

There were no statistically significant increases in the proportion of aberrant cells, in the presence of S-9 mix, at the 18 or 32 hour harvests. In the absence of S-9 mix, in the second test at the 18 hour harvest, there was a statistically significant increase in the number of aberrant cells at the highest concentration, 30 ug/ml, when gap damage was excluded. However, this increase lies within the historical control range, there was no dose-relationship and this increase was not seen in the first test or at the later sampling time. Therefore, it is concluded that this increase is not treatment-related. All positive control compounds caused large, statistically significant increases in the proportion of aberrant cells. It is concluded that OTNE has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.

In vitro gene mutation in mammalian cells

In an in vitro mammalian cell gene mutation test which was performed in accordance with OECD TG 476 and under GLP conditions, the mutagenic properties of TM 09-221 were determined in mouse lymphoma cells. Based on the preliminary toxicity test, the mutation tests were performed using the following concentrations: Test 1: without S9 mix (3 hours) 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35 and 40 µg/mL, with S9 mix (3 hours) 10, 30, 50, 70, 90, 110, 130, 150 and 200 µg/mL, Test 2: without S9 mix (24 hours) 10, 20, 30, 40, 50, 60, 70 and 80 µg/mL. Exposure to solvent ethanol was included as a negative control, and methylmethanesulfate and benzo(a)pyrene were included as positive control substances.

Following 3 hour treatment in the absence and presence of S9 mix, there were no increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor (GEF), within acceptable levels of toxicity. The maximum concentrations assessed for mutant frequency in the 3 hour treatment in the absence and presence of S9 mix were 40 and 90 µg/mL respectively. In the absence and presence of S9 mix RTG was reduced to 20 and 23% respectively. In the 24 hour treatment, the maximum concentration assessed for mutant frequency was 50 µg/mL. No increase in mutant frequency exceeded the sum of the mean concurrent vehicle control mutant frequency and the GEF was observed and RTG was reduced to 16%. It was concluded that OTNE did not demonstrate mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described.

In vivo micronucleus test - rat (OECD TG 474)

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.

In vivo micronucleus test - mouse (OECD TG 474)

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.

Justification for classification or non-classification

Based on the available information in the dossier, OTNE does not need to be classified for genotoxicity when considering the criteria outlined in EU CLP (1272/2008/EC and its amendments).