(3R)-1,1,3-trimethyl-2,3-dihydro-1H-inden-4-amine

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Nov 2014 - 14 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Nov 2014 - 14 Apr 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 26 Sep 2014

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certifying authority: Food Safety and Consumers Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Remarks:

RccHan:WIST

Details on species / strain selection:

RccHan:WIST rats are commonly used for safety assessment studies like the comet assay and the lab has accumulated background information.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hannou Breeding Center C-building, Japan Laboratory Animals, Inc., Saitama, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 252.8 g (males)
- Housing: Up to 5 rats were housed together in plastic cages (W290 x D340 x H170 mm, Crea Japan Inc., Tokyo, Japan) with shavings (Whiteflake, Charles River Laboratories Japan, Inc.).
- Diet: laboratory animal chow (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan), ad libitum
- Water: tap water via an automatic water supply system (Labo Engineering Co., Ltd., Shizuoka, Japan), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25 (actual temperature: 22.7 - 23.9)
- Humidity (%): 40 - 70 (actual humidity: 44.6 - 69.4)
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of vehicle: The vehicle was chosen for its common use in oral administration with relative non-toxicity to the animals and non-mutagenicity.
- Concentration of test material in vehicle: 4.80 - 4.85 mg/mL for the nominal 5 mg/mL dose; 197 - 201 mg/mL for the nominal 200 mg/mL dose
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: V3T2547

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was soluble in corn oil up to 200 mg/mL. The test substance was prepared for dosing as a weight-to-volume mixture in corn oil and mixed evenly by turning the container upside down. The dosing solution was protected from light and stored at room temperature until use. The test substance was stable at 5 - 200 mg/mL in corn oil for 24 h at room temperature.

DETAILS ON DOSING
The dose volume was 10 mL/kg. The dosing mixtures were administered using a syringe (Terumo Corporation, Tokyo, Japan) affixed to an elastic catheter (Terumo Corporation, Tokyo, Japan). The individual dose volumes were calculated based on body weights taken just before administration. Dosing mixtures of the test substance and the positive control were prepared at times of use and administration was completed within 4 h after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

once daily

Post exposure period:

Sampling time point: 3 h after the final dosing

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

- Ethyl Methanesulfonate (EMS)
- Justification for choice of positive control(s): EMS was expected to demonstrate the sensitivity of the test system.
- Route of administration: oral gavage
- Preparation: dissolved in saline at 20 mg/mL
- Dose: 200 mg/kg bw/day
- Dosing volume: 10 mL/kg
- Administration: once daily for 2 consecutive days

Tissues and cell types examined:

liver, stomach and spleen

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary acute toxicity test was performed to find the maximum tolerated dose. This dose was found to be 800 mg/kg bw/day which was chosen as the top dose for the main study.

TREATMENT AND SAMPLING TIMES: Rats were euthanized by carbon dioxide inhalation 3 h after the final dosing, and liver, stomach and spleen removed. The stomach was cut open longitudinally and rinsed with the cold homogenizing buffer. The liver (left lateral lobe), the glandular stomach and the spleen were collected from all animals at gross pathology and fixed in neutral phosphate buffered 10% formaldehyde solution.

DETAILS OF SLIDE PREPARATION:
Portions (5-mm square) of the liver (left lateral lobe) and spleen were minced with a pair of scissors and suspended in 5 mL of ice-cold homogenizing buffer. The cell suspensions were diluted using ice-cold homogenizing buffer after filtration by a 100 µm mesh (Corning, Inc., USA). The glandular stomach was placed into ice-cold homogenizing buffer and incubated on ice for 15 - 30 min. After incubation, the surface epithelia were gently scraped a few times using a cell scraper (IWAKI, Asahi glass Co., Ltd., Japan) and washed out with ice-cold homogenizing buffer, and the washings were discarded. After that, the lower layer of the epithelia was scraped by a metal spatula to release the cells and the cells were suspended in 5 mL of ice-cold homogenizing buffer. The cell suspension was diluted using ice-cold homogenizing buffer after filtration by a 40 µm mesh (Corning, Inc., USA). Each cell suspension was mixed with 0.5% of low melting agarose solution in the ratio 1:9 and the mixture was placed at 0.15 mL/well on a MAS coated slide glass (Matsunami glass Ind., Ltd., Japan) and solidified on ice. Each slide was placed in lysing buffer overnight in a refrigerator set at 0 - 10 °C. Slides were rinsed with purified water and placed randomly in a submarine-type electrophoresis chamber (NB-1012, Nihon Eido Co., Ltd., Japan). Cold electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH > 13) was gently poured into the chamber until the slides were completely immersed. After 20 min, electrophoresis was conducted using power supply (PowerPack Basic, BIORAD Laboratories, Inc., USA) at a constant voltage of 0.7 V/cm (26 V) at approximately 300 mA for 20 minutes. The temperature of the electrophoresis buffer was set at 2 - 10 °C. After electrophoresis, the specimens were immersed into neutralization buffer (0.4 M Tris (hydroxymethyl) aminomethane, pH 7.5) for at least 5 min, dehydrated with ethanol (≥ 99.5%) and air dried. The slides were coded in each organ and analyzed in a blind manner. Approximately 100 µL of staining solution (SYBR® Gold nucleic acid gel stain, Invitrogen, USA) diluted 10000-fold with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) was dropped on the slide just before observation. The slides were stained for 15 - 30 min and a coverslip applied.

COMPOSITION OF BUFFERS USED
- Homogenizing buffer (per 1000 mL):
x10 Hanks' balanced salt solution (Gibco, Life Technologies Corp., USA): 100 mL; EDTA*2Na*2H2O: 7.4 g; Dimethyl sulfoxide (DMSO): 100 mL
The above reagents except DMSO were dissolved in purified water and adjusted to a pH of 7.5 with sodium hydroxide. The solution was diluted to 900 mL with purified water. DMSO was added to the solution just before use.
- 0.5% Agarose solution (per 100 mL):
NuSieve GTG Agarose (Lot No.0000437528, Lonza Corporation, USA): 0.5 g; Dulbecco's phosphate-buffered saline: 100 mL
Dulbecco's phosphate-buffered saline was prepared from Dulbecco's PBS (Lot No. 184301, Nissui pharmaceutical Co., Ltd., Japan). Dulbecco's PBS (4.8 g) was dissolved in 500 mL of purified water and then autoclaved. The above reagents were mixed, dissolved by heating in a microwave oven and maintained at 37 - 45 °C until use.
- Lysing buffer (per 1000 mL):
NaCl: 146.1g; EDTA*2Na*2H2O: 37.2 g; Tris (hydroxymethyl) aminomethane: 1.2 g; NaOH: 8.0g; DMSO: 100 mL; Triton-X100: 10 mL
The above reagents except DMSO and Triton X-100 were dissolved in purified water and adjusted to a pH of 10.0 with sodium hydroxide. The solution was diluted to 890 mL with purified water. DMSO and Triton X-100 were added to the solution just before use.


METHOD OF ANALYSIS:
The images of DNA migration of cells were examined using a fluorescence microscope with IB excitation. The images were imported to the computer through a CCD camera attached on the microscope and analyzed by a Comet assay analyzer (Comet Assay IV system, Perceptive Instruments, UK). 75 cells per slide were observed and 2 slides per animal were examined in each organ. After the median of % tail DNA was obtained in each slide, the mean of median from 2 slides was calculated as the value of % tail DNA of individual animals. By using fluorescence microscope (IB excitation) with a 20-fold objective lens and 10-fold eye lens in a dry system, the number of hedgehogs was counted. The mean of the frequency of hedgehogs from 2 slides was calculated.

Evaluation criteria:

EVALUATION CRITERIA:
The test substance is judged to induce DNA damage if the following criteria are satisfied:
a) there is a statistically significant increase in the % tail DNA in at least one dose group when compared with the concurrent vehicle control value,
b) the increase is statistically significant dose-related, and
c) the % tail DNA in increased dose group exceeds the acceptable negative (vehicle) control range (mean ± 2SD, calculated from the historical control data, see table 2).

When the test substance is judged to be positive, the histopathological examination is performed. Based on the results of the histopathological findings and the frequency of hedgehogs in each organ, the cell toxicity and its biological relevance are taken into consideration when making the final judgement.

Statistics:

STATISTICS:
The % tail DNA of individual animal was used as parameters for statistical processing to evaluate the DNA damage. The % tail DNA was analyzed by Dunnett multiple comparison test between the vehicle control group and each dose group. The level of significance was tested at p = 0.05 (two tailed). The Student’s t-test was used for the comparison of the vehicle control group with the positive control group. The level of significance was tested at p = 0.025 (one tailed). When the test substance induces a statistically significant increase in the % tail DNA, the dose-response relation is tested with the linear regression analysis. The level of significance is tested at p = 0.025 (one tailed).

The t-test was used for analysis of the animal body weight changes. The Student's t-test was used if p ≥ 0.05 in the F-test and the Welch's t-test was used if p < 0.05 in the F-test. The levels of significance were 0.04 and 0.01 (two-tailed).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

800 mg/kg: marked clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: liver tissue

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

800 mg/kg: marked clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: stomach tissue

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

800 mg/kg: marked clinical signs of toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: spleen tissue

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw/day (doses tested: 500, 1000 and 2000 mg/kg bw/day for 3 consecutive days in 5 animals/sex/dose)
- Solubility: test substance soluble in corn oil
- Clinical signs of toxicity in test animals: 2000 mg/kg bw/day: Prone position, ataxic gait and urinary incontinence (male only) were observed in both sexes within 4 h after 1st dosing. Just before 2nd dosing, 1 out of 5 animals for each sex were found dead, and remaining animals were euthanized because severe clinical signs, such as prone position and dyspnea were observed in both sexes.
1000 mg/kg bw/day: In male rats, ataxic gait, decrease of spontaneous activity and urinary incontinence were observed after 1st and 2nd dosing. After 3rd dosing, one out of five males was found dead and severe clinical signs such as prone position, ataxic gait and urinary incontinence were observe in remaining male animals. In female rats, ataxic gait, prone position, dyspnea, decrease of spontaneous activity and urinary incontinence were observed after 1st dosing. After 2nd dosing, prone position, ataxic gait and urinary incontinence were observed within 4 h and 4 out of 5 females were found dead before 3rd dosing. The remaining 1 female was euthanized because a severe clinical sign (prone position) was observed.
500 mg/kg bw/day: Although no death was observed in both sexes, ataxic gait was observed in females within 4 h after 1st and 2nd dosing.
- Effects on body weight: A statistically significant decrease in the body weight gains was observed in both sexes at 2000 and 1000 mg/kg bw/day after 1st dosing and in males at 500 mg/kg bw/day after 2nd dosing. No statistically significant decrease in the body weight gains was observed in the female rats at 500 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY
- Clinical signs: Although no death was observed at the dose level of 800 mg/kg bw/day, marked clinical signs such as ataxic gait, urinary incontinence and soft stool were observed. Therefore, the maximum dose of 800 mg/kg bw/day was verified as the maximum tolerable dose, and it is considered that the test substance reached general circulation and target tissues up to sufficient high dose levels. No abnormal clinical sign was observed at dose levels of 400 and 200 mg/kg bw/day of test substance groups and positive control group.
- Body weight: A statistically significant decrease in the body weight gains was observed in all the test substance groups just before 3rd dosing. The significance levels were < 0.01 in 800 mg/kg bw/day dosing group and < 0.05 in 400 and 200 mg/kg bw/day dosing groups, respectively. These depressions of body weight and body weight gain also indicated that the dose setting of the main assay was appropriate. In the positive control group, a statistically significant decrease was observed just before 2nd dosing.
- Gross pathology: No abnormal findings were observed in both the test substance groups and positive control groups.
-% tail DNA (liver): In the vehicle control group, group mean of % tail DNA was 0.162% and fell within the acceptance negative (vehicle) control range (the mean ± 2SD of the historical control was 0.145 ± 0.131% and the mean ± 2SD was set as the acceptable control range). In the EMS-treated positive control group, the group mean of % tail DNA was 4.61% and a statistically significant increase compared to the vehicle control group was observed. Thus, the test was judged to be acceptable.
The group means of % tail DNA at the dose levels of 200, 400 and 800 mg/kg bw/day were 0.137%, 0.127% and 0.193%, respectively, and the frequencies of hedgehogs were 0.7%, 0.5% and 0.9%, respectively. No statistically significant increase in the % tail DNA, nor in the frequency of hedgehogs, compared to the vehicle control group (0.8%), was observed at any of the doses.
-% tail DNA (stomach): In the vehicle control group, group mean of % tail DNA was 1.49% and fell within the acceptance negative (vehicle) control range (the mean ± 2SD of the historical control was 3.17 ± 2.19% and the mean ± 2SD was set as the acceptable control range). In the EMS-treated positive control group, the group mean of % tail DNA was 12.6% and a statistically significant increase compared to the vehicle control group was observed. Thus, the test was judged to be acceptable.
The group means of % tail DNA at the dose levels of 200, 400 and 800 mg/kg bw/day were 1.22%, 1.61% and 1.21%, respectively, and the frequencies of hedgehogs were 2.8%, 3.7% and 2.8%, respectively. No statistically significant increase in the % tail DNA, nor in the frequency of hedgehogs, compared to the vehicle control group (4.1%), was observed at any of the doses.
-% tail DNA (spleen): In the vehicle control group, group mean of % tail DNA was 0.117% and fell within the acceptance negative (vehicle) control range (the mean ± 2SD of the historical control was 0.0642 ± 0.0452% and the mean ± 2 SD was set as the acceptable control range). In the EMS-treated positive control group, the group mean of % tail DNA was 1.45% and a statistically significant increase compared to the vehicle control group was observed. Thus, the test was judged to be acceptable.
The group means of % tail DNA at the dose levels of 200, 400 and 800 mg/kg bw/day were 0.125%, 0.0925% and 0.118%, respectively, and the frequencies of hedgehogs were 0.5%, 0.3% and 0.4%, respectively. No statistically significant increase in the % tail DNA, nor in the frequency of hedgehogs, compared to the vehicle control group (0.3%), was observed at any of the doses.

Table 1: Results of the in vivo Comet Assay in male animals

Exposure group	Dose (mg/kg bw/day)	Organ	Treatment period^a) (h)	No. of animals	Total number of cells analyzed	% tail DNA (Mean ± SD)	Frequency of hedgehogs (%) (Mean ± SD)
Corn oil	-	Liver	48, 24, 3	5	750	0.162 ± 0.0367	0.8 ± 0.56
Test substance	200		48, 24, 3	5	750	0.137 ± 0.0393	0.7 ± 0.82
	400		48, 24, 3	5	750	0.127 ± 0.0334	0.5 ± 0.56
	800		48, 24, 3	5	750	0.193 ± 0.0246	0.9 ± 0.76
EMS	200		24, 3	5	750	4.61 ± 1.61 *	1.2 ± 0.99
Corn oil	-	Stomach	48, 24, 3	5	750	1.49 ± 0.669	4.1 ± 3.21
Test substance	200		48, 24, 3	5	750	1.22 ± 0.681	2.8 ± 1.28
	400		48, 24, 3	5	750	1.61 ± 0.477	3.7 ± 1.46
	800		48, 24, 3	5	750	1.21 ± 0.607	2.8 ± 1.45
EMS	200		24, 3	5	750	12.6 ± 3.90 *	5.9 ± 3.31
Corn oil	-	Spleen	48, 24, 3	5	750	0.117 ± 0.0223	0.3 ± 0.37
Test substance	200		48, 24, 3	5	750	0.125 ± 0.0397	0.5 ± 0.87
	400		48, 24, 3	5	750	0.0925 ± 0.0147	0.3 ± 0.37
	800		48, 24, 3	5	750	0.118 ± 0.0407	0.4 ± 0.60
EMS	200		24, 3	5	750	1.45 ± 0.994 *	0.5 ± 0.56

EMS = Ethyl Methanesulfonate

^a)Treatment period between each administration and tissue sampling

*p < 0.025 (t-test, one-tailed)

 

 

 

Table 2: Historical control data (2009 - 2014) from Crl:CD (SD) and RccHan:WIST rats

 

	Organ	Total number of groups	% tail intensity with comet assay (%, Mean ± SD)
Negative (vehicle) control	liver	10	0.145 ± 0.131
	stomach	6	3.17 ± 2.19
	spleen	5	0.0642 ± 0.0452
Positive control (EMS, 200 mg/kg bw/day, oral, 24 and 3 h treatment)	liver	7	8.94 ± 3.17
	stomach	6	22.8 ± 6.22
	spleen	2	5.56 ± 2.21

 

Conclusions:

It is concluded that (3R-)2,3-dihydro-1,1,3-trimethyl-1H-inden-4amine does not induce DNA damage in the liver, stomach or spleen.
As this test, in combination with the available in vivo micronucleus assay, is a higher tier follow-up on the positive in vitro Ames test, the general conclusion on the substance's genotoxic potential is negative.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 26 September 2014

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted 29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Certifying authority: Food Safety and Consumers Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

(3R-)1,1,3-trimethylindan-4-amine

EC Number:

836-285-0

Cas Number:

125349-37-3

Molecular formula:

C12H17N

IUPAC Name:

(3R-)1,1,3-trimethylindan-4-amine

Test animals

Species:

rat

Strain:

Wistar

Remarks:

RccHan:WIST

Details on species / strain selection:

RccHan:WIST rats are are commonly used for safety assessment studies like the micronucleus test and the lab has accumulated background information.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hannou Breeding Center C-building, Japan Laboratory Animals, Inc., Saitama, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 252.8 g (males)
- Housing: Up to 5 rats were housed together in plastic cages (W290 x D340 x H170 mm, Crea Japan Inc., Tokyo, Japan) with shavings (Whiteflake, Charles River Laboratories Japan, Inc.).
- Diet: laboratory animal chow (CRF-1, Oriental Yeast Co. Ltd., Tokyo, Japan), ad libitum
- Water: tap water via an automatic water supply system (Labo Engineering Co., Ltd., Shizuoka, Japan), ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 25 (actual temperature: 22.7 - 23.9)
- Humidity (%): 40 - 70 (actual humidity: 44.6 - 69.4)
- Air changes (per hr): ≥ 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of vehicle: The vehicle was chosen for its common use in oral administration with relative non-toxicity to the animals and non-mutagenicity.
- Concentration of test material in vehicle: 4.80 - 4.85 mg/mL for the nominal 5 mg/mL dose; 197 - 201 mg/mL for the nominal 200 mg/mL dose
- Amount of vehicle: 10 mL/kg
- Lot/batch no.: V3T2547

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test substance was soluble in corn oil up to 200 mg/mL. The test substance was prepared for dosing as a weight-to-volume mixture in corn oil and mixed evenly by turning the container upside down. The dosing solution was protected from light and stored at room temperature until use. The test substance was stable at 5 - 200 mg/mL in corn oil for 24 h at room temperature.

DETAILS ON DOSING
The dose volume was 10 mL/kg. The dosing mixtures were administered using a syringe (Terumo Corporation, Tokyo, Japan) affixed to an elastic catheter (Terumo Corporation, Tokyo, Japan). The individual dose volumes were calculated based on body weights taken just before administration. Dosing mixtures of the test substance and the positive control were prepared at times of use and administration was completed within 4 h after preparation.

Duration of treatment / exposure:

3 consecutive days

Frequency of treatment:

once daily

Post exposure period:

Sampling time point: 3 h after the final dosing

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

800 mg/kg bw/day (actual dose received)

Remarks:

Maximum tolerable dose

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

- Ethyl Methanesulfonate (EMS)
- Justification for choice of positive control(s): EMS was expected to demonstrate the sensitivity of the test system.
- Route of administration: oral gavage
- Preparation: dissolved in saline at 20 mg/mL
- Dose: 200 mg/kg bw/day
- Dosing volume: 10 mL/kg bw
- Administration: once daily for 2 consecutive days

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary acute toxicity test was performed in male and female rats to find the maximum tolerated dose. This dose was found to be 800 mg/kg bw/day which was chosen as the top dose for the main study.

TREATMENT AND BONE MARROW SAMPLING:
Rats were euthanized by carbon dioxide inhalation 3 h after the final dosing and the femur was removed. Bone marrow cells were washed out from the femurs with 1.0 mL fetal bovine serum containing 25 mM EDTA for each femur using a 1 mL syringe.

MICRONUCLEUS SLIDE PREPARATION:
The cell suspensions were centrifuged (1000 rpm, 5 min, at room temperature) to give cell pellets. After decantation, each of the cell pellets was suspended in the residual serum. The cell suspension was diluted arbitrarily with fetal bovine serum (containing 25 mM EDTA) and a small portion was smeared on a glass slide. The slides were fixed with methanol for 5 min within one day after preparation.

METHOD OF ANALYSIS:
The slides were coded and analyzed in a blind manner. Just before observation, a drop of acridine orange at 40 µg/mL was put on the fixed smears, and the slides were covered with cover-slips. Observation was performed with a fluorescence microscope (B excitation) using a 40-fold objective lens and 15-fold eye lens in a dry system. Erythrocytes with particles which emitted yellowish green fluorescence like nuclei and sharply outlined were identified as micronucleated erythrocytes. The staining procedure permitted the differentiation by color of polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs) (red fluorescent and non-fluorescent, respectively). The incidence of micronucleated cells in 4000 PCEs was scored for each animal, and the incidence of PCEs in 1000 erythrocytes (PCEs and NCEs) was also examined.

Evaluation criteria:

EVALUATION CRITERIA:
The test substance is judged to induce micronuclei if the following criteria are satisfied:
a) there is a statistically significant increase in the incidence of micronucleated PCEs in at least one dose group when compared with the concurrent vehicle control value,
b) the increase is statistically significant dose-related, and
c) the incidence of micronucleated PCEs in the increased dose group exceeds the acceptable negative (vehicle) control range (mean ± 2SD, calculated from the historical control data, see table 2).

Statistics:

STATISTICS:
Statistical analysis for the incidence of micronucleated PCE cells was performed using the Kastenbaum-Bowman tables which were based on the binomial distribution. The levels of significance were tested at p = 0.05 and 0.01. When the test substance induces a statistically significant increase in the incidence of micronucleated PCEs, the dose-response relation is tested with the Cochran-Armitage trend test. The levels of significance are tested at p = 0.05 and 0.01 (one-tailed). The t-test was used for analysis of the ratio of PCEs to total erythrocytes. The Student's t-test was used if p ≥ 0.05 in the F-test and the Welch's t-test was used if p < 0.05 in the F-test. The levels of significance were 0.04 and 0.01 (two-tailed).

The t-test was used for analysis of the animal body weight changes. The Student's t-test was used if p ≥ 0.05 in the F-test and the Welch's t-test was used if p < 0.05 in the F-test. The levels of significance were 0.04 and 0.01 (two-tailed).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

800 mg/kg bw/day: bone marrow toxicity

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 500 - 2000 mg/kg bw/day (doses tested: 500, 1000 and 2000 mg/kg bw/day for 3 consecutive days in 5 animals/sex/dose)
- Solubility: test substance soluble in corn oil
- Clinical signs of toxicity in test animals: 2000 mg/kg bw/day: Prone position, ataxic gait and urinary incontinence (male only) were observed in both sexes within 4 h after 1st dosing. Just before 2nd dosing, 1 out of 5 animals for each sex were found dead, and remaining animals were euthanized because severe clinical signs, such as prone position and dyspnea were observed in both sexes.
1000 mg/kg bw/day: In male rats, ataxic gait, decrease of spontaneous activity and urinary incontinence were observed after 1st and 2nd dosing. After 3rd dosing, one out of five males was found dead and severe clinical signs such as prone position, ataxic gait and urinary incontinence were observe in remaining male animals. In female rats, ataxic gait, prone position, dyspnea, decrease of spontaneous activity and urinary incontinence were observed after 1st dosing. After 2nd dosing, prone position, ataxic gait and urinary incontinence were observed within 4 h and 4 out of 5 females were found dead before 3rd dosing. The remaining 1 female was euthanized because a severe clinical sign (prone position) was observed.
500 mg/kg bw/day: Although no death was observed in both sexes, ataxic gait was observed in females within 4 h after 1st and 2nd dosing.
- Effects on body weight: A statistically significant decrease in the body weight gains was observed in both sexes at 2000 and 1000 mg/kg bw/day after 1st dosing and in males at 500 mg/kg bw/day after 2nd dosing. No statistically significant decrease in the body weight gains was observed in the female rats at 500 mg/kg bw/day.

RESULTS OF DEFINITIVE STUDY (see table 1)
- Clinical signs: Although no death was observed at the dose level of 800 mg/kg bw/day, marked clinical signs such as ataxic gait, urinary incontinence and soft stool were observed. Therefore, the maximum dose of 800 mg/kg bw/day was verified as the maximum tolerable dose, and it is considered that the test substance reached general circulation and target tissues up to sufficient high dose levels. No abnormal clinical sign was observed at dose levels of 400 and 200 mg/kg bw/day of test substance groups and positive control group.
- Body weight: A statistically significant decrease in the body weight gains was observed in all the test substance groups just before 3rd dosing. The significance levels were < 0.01 in 800 mg/kg bw/day dosing group and < 0.05 in 400 and 200 mg/kg bw/day dosing groups, respectively. These depressions of body weight and body weight gain also indicated that the dose setting of the main assay was appropriate. In the positive control group, a statistically significant decrease was observed just before 2nd dosing.
- Acceptability of the test: In the vehicle control group, the group mean of micronucleated PCEs was 0.12% and fell within the acceptance negative (vehicle) control range (the mean ± 2SD of the historical control was 0.20 ± 0.06% and the mean ± 2SD was set as the acceptable control range). In the EMS-treated positive control group, the group mean of micronucleated PCEs was 1.47% and a statistically significant increase compared to the vehicle control group was observed. Thus, the test was judged to be acceptable.
- Induction of micronuclei: The mean of micronucleated PCEs was 0.16%, 0.17%, 0.13% at the dose levels of 200, 400 and 800 mg/kg bw/day, respectively. The test substance induced no significant increase in the incidence of PCEs.
- Ratio of PCE/NCE: A statistically significant decrease in the ratio of PCEs to whole erythrocytes was observed at 800 mg/kg bw/day when compared with the concurrent vehicle control. Therefore, it was considered that the effect was caused by the test substance which reached bone marrow cells.
- Appropriateness of dose levels and route: Based on the results of the preliminary study, it was considered that there was no substantial difference between sexes in toxicity and 800 mg/kg bw/day was considered to be the maximum tolerable dose in each sex. Since there was no substantial difference between sexes in toxicity, male rats, which are extensively validated in the comet assay, were selected for the main study. The route of the test substance administration was oral by gavage. This is a plausible route of exposure in humans and is the standard in the comet assay and micronucleus test.

Any other information on results incl. tables

Table 1: Results of the in vivo Micronucleus Assay in male animals

Exposure group	Dose (mg/kg bw/day)	Treatment period^a) (h)	No. of animals	PCE/(PCE+NCE)^b) (% Mean ± SD)	PCE with micronuclei
					(%, Mean ± SD)	[range]
Vehicle control (corn oil)	-	48, 24, 3	5	77.4 ± 5.98	0.12 ± 0.029	[3 - 6] / 4000
Positive control (EMS)	200	24, 3	5	75.0 ± 6.10	1.47 ± 0.461 **	[41 - 89] / 4000
Test substance	200	48, 24, 3	5	76.8 ± 6.20	0.16 ± 0.058	[4 - 10] / 4000
Test substance	400	48, 24, 3	5	74.2 ± 13.50	0.17 ± 0.074	[4 - 11] / 4000
Test substance	800	48, 24, 3	5	66.4 ± 4.88 *	0.13 ± 0.045	[3 - 7] / 4000

PCE = Polychromatic Erythrocytes; NCE = Normochromatic Erythrocytes; EMS = Ethyl Methanesulfonate

^a)Treatment period between each administration and tissue sampling

^b)1000 erythroctes were analysed from each animal

*p < 0.05 (t-test, two-tailed); **p < 0.01 (Kastenbaum-Bowman)

 

 

Table 2: Historical control data (2007 - 2014)

 

	Strain, Sex	Total number of groups	PCE with micronuclei (%, Mean ± SD)
Negative (vehicle) control	RccHan:WIST, male	7	0.20 ± 0.06
Positive control (EMS, 200 mg/kg bw/day, oral, 24 and 3 h treatment)	RccHan:WIST, male	1	1.36

 

 

Applicant's summary and conclusion

Conclusions:

It is concluded that (3R-)2,3-dihydro-1,1,3-trimethyl-1H-inden-4amine has no potential to induce micronuclei in rat bone marrow cells under the conditions tested.
As this test, in combination with the available in vivo comet assay, is a higher tier follow-up on the positive in vitro Ames test, the general conclusion on the substance's genotoxic potential is negative.