Benzeneacetic acid, 4-[4-[4-(hydroxydiphenylmethyl)-1-piperidinyl]-1-butyn-1-yl]-.alpha.,.alpha.-dimethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Three studies on genetic toxicity were performed: (i) Ames test (OECD 471), (ii) Chromosome Aberration Test (OECD 473) and (iii) Micronucleus Assay in Bone Marrow Cells (OECD 474). FEX0-07 in vitro: - caused gene mutations by base pair changes in the genome of the tester strain TA 100 in Ames Test. Therefore, FEX0-07 was considered to be mutagenic in this bacterial reverse mutation assay - was considered to be clastogenic in this chromosome aberration test in V79 cells (Chinese hamster cell fine) in the presence of metabolic activation (S9 mix). FEXO-07 in vivo: - did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, Guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, Annex 4C, dated May 19, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH-Harmonised T ripartite Guideline S 2 A: "Genotoxicity: Guidance on Specific Aspects of Regulatory Genotoxicity Tests of Pharmaceuticals" (CPMP/ICH/141/95), dated Aprii 1996.

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH-Harmonised Tripartite Guideline S 2 B: "Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals" (CPMP/ICH/17 4/95), dated July 1997.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Number of animals: 72 (36 males/36 females)
lnitial age at start of acclimatisation: 7- 10 weeks
Acclimatisation: minimum 5 days
lnitial Body Weight at start of treatment:
- males mean value 36.7 g (SD± 1.7 g)
- females mean value 28.4 g (SD± 2.0 g)

The animals were under quarantine in the animal house of RCC - CCR for a minimum of five days after their arrival.
During this period the animals did not show any signs of illness or altered behaviour.
The test animals were distributed into the test groups at random and identified by cage number.

Housing: single
Cage type: Makrolon Type l, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature: 22 ± 3 °C; relative humidity: 30 - 70 %; artificial light: 6.00 a.m. - 6.00 p.m.

Route of administration:

oral: unspecified

Vehicle:

Corn oil
(On the day of the experiment, the test item was formulated in corn oil. The vehicle was chosen to its relative non-toxicity for the animals.).

Details on exposure:

All animals will receive a single standard volume orally.

Volume administered (vehicle control and positive control): 10 mL/kg b.w.

Duration of treatment / exposure:

48 h

Frequency of treatment:

Frequency of administration (vehicle control): once

Remarks:

Doses / Concentrations:
2000 mg/kg b.w.
Basis:
no data
On the basis of pre-experiment for toxicity data 2000 mg/kg b.w. was estimated to be suitable

Remarks:

Doses / Concentrations:
1000 mg/kg b.w.
Basis:
no data
For the mid dose group [12 animals (6 males, 6 females)] each received orally this single dose

Remarks:

Doses / Concentrations:
500 mg/kg b.w.
Basis:
no data

No. of animals per sex per dose:

6

[Ten animals (5 males, 5 females) per test group were evaluated as described below. The remaining 6th animal of each sex in the respective test group test group is usually evaluated in case an animal dies in its test group spontaneously].

Control animals:

yes

Positive control(s):

Name: CPA; Cyclophosphamide
Dissolved in: deionised water
Dosing: 40 mg/kg b.w.

( Solution prepared on day of administration; the stability of CPA at room temperature was judged as sufficient).

Details of tissue and slide preparation:

Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives.
At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei.
To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data
_ the positive controls are in the range of our historical control data
- at least 4 animals per group and sex can be evaluated
- PCE to erythrocyte ratio should not be less than 20 % of the negative control

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results.
However, the primary point of consideration is the biological relevance of the results.

Additional information on results:

None of the animals treated with 1000 or 500 mg/kg b.w. test item expressed any toxic reactions, nor those treated with the vehicle control (corn oil).

The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that FEX0-07 did not have any cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item.
The mean values of micronuclei observed after treatment with FEX0-07 were near to the value of the vehicle control group.

40 mg/kg b. w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

For the table of results, see the file attached below.

Conclusions:

Interpretation of results (migrated information): negative
It can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.
FEX0-07 is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

The study was performed to investigate the potential of FEX0-07 to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was formulated in corn oil, which was also used as vehicle control.

The volume administered orally was 20 mL/kg. The volume of the positive control administered was 10 mL/kg b.w. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

- 24 h preparation interval: 500, 1000, and 2000 mg/kg b.w

- 48 h preparation interval: 2000 mg/kg b. w .

The highest dose (2000 mg/kg; maximum guideline-recommended dose) was estimated by a pre-experiment to be suitable. After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that FEX0-07 did not exert any cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

40 mg/kg b. w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

In vitro tests

1) Reverse Mutation Assay using Bacteria (Salmonella typhimurium)

One study (Klimisch score=1) was identified, in which FEXO-07 was tested to investigate the potential its to induce gene mutations; the plate incorporation test was performed with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 using a method according to OECD 471, EU method B.13/14 and EPA OPPTS 870.5100.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation.

In experiment I toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (with and without

metabolic activation). In experiment II toxic effects of the test item were noted at concentrations of 750 µg/plate and higher (with and without metabolic activation).

FEX0-07 caused gene mutations by base pair changes in the genome of the tester strain TA 100. Therefore, FEX0-07 is considered to be mutagenic in this bacterial reverse mutation assay.

2) In vitro Chromosome Aberration Test in Chinese Hamster V79 Cells

One study (Klimisch score=1) was identified, in which FEXO-07 was tested to investigate its potential to induce chromosome aberrations in V79 cells of the Chinese hamster in vitro with and without metabolic activation.

Test item concentrations between 2.7 and 350 µg/ml (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the presence of s9 mix, precipitation of the test item after 4 hrs treatment was observed at 350 µg/ml.

FEXO-07 induced structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cellline) in vitro. Therefore, the substance was considered to be clastogenic in this chromosome aberration test in the presence of S9 tmix.

In vivo test

1) Micronucleus Assay in Bone Marrow Cells of the Mouse

One study (Klimisch score=1) was identified, in which FEXO-07 was tested to investigate its potential to induce micronuclei in polychromatic erythrocytes in the bone marrow of the mouse. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 20 mL/kg. The following dose levels of the test item were investigated: (i) 24 h preparation interval: 500, 1000, and 2000 mg/kg bw and (ii) 48 h preparation interval: 2000 mg/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.

FEXO-07 did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, it was considered to be non-mutagenic in this micronucleus assay.

Justification for selection of genetic toxicity endpoint
The selected test has been performed in vivo; therefore, it was deemed as more reliable than the test carried out in vitro.

Justification for classification or non-classification

Based on evaluation of the genetic toxicity data (in vitro and in vivo) discussed above, FEXO-07 does not complitely meet the criteria for classification and labelling under both the CLP Regulation and the Directive 67/548/EEC: the results were judged as ambiguous to assign a classification and labelling to the substance.