Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
The mutagenic potential of benoxacor has been assessed in a number of core genotoxicity assays in vivo and in vitro, covering a range of endpoints including gene mutation, DNA repair and clastogenicity. The data indicate the test material is not genotoxic.
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 March 1986 to 22 September 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Two part main experiment: In the first part a single dose of 5000 mg/kg was administered to 3 groups of 8 male and 8 female Chinese hamsters. The groups were killed after 16, 24, and 48 hours. Three same sized negative control groups were treated with the vehicle and also killed after 16, 24, or 48 hours. The sensitivity of the test system was checked by treating one further group with 64 mg/kg of the known mutagen Cyclophosphamide (CPA). The animals of this group were killed 24 hours after treatment. Analytical purity: 93.9%

In the second part three groups of 8 male and 8 female hamsters were treated with a single dose of 1250, 2500, or 5000 mg/kg of CGA 154281 and killed after 24 hours. Negative and positive control groups were used. After sacrifice the bone marrow from the shafts of both femurs was removed and smears were prepared on slides. Routinely the slides of five animals/sex/dose were stained with May-Grunwald solution and scored for micronuclei. As an exception, the slides from 3 males and 7 females which were killed after 16 hours (part 1) were scored, since only three males in each group furnished well differentiated cells. From each animal the ratio of polychromatic to normochromatic erythrocytes was determined as an indicator for toxic effects on the erythropoiesis and 1000 polychromatic erythrocytes were scored for micronuclei.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
hamster, Chinese
Strain:
other: Random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
3 groups each consisting of 8 male and 8 female Chinese hamsters.
TEST ANIMALS
- Age at study initiation: females 6-10 weeks, males 4-9 weeks
- Weight at study initiation: females 22-32 g, males 20-35 g
- Diet: NAFAG No.924
- Water: Tap water ad libitum
- Acclimation period: 4-5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22-23°C
- Humidity: 52-59%
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 03 March 1986 To: 22 September 1986
Route of administration:
oral: gavage
Vehicle:
0.5% aqueous carboxymethylcellulose (CMC).
Details on exposure:
Part 1: The different groups of animals were killed after 16, 24, and 48 hours.
Part 2: The different groups of animals were killed after 24 hours.
Duration of treatment / exposure:
16 to 48 hours
Frequency of treatment:
Single dose
Post exposure period:
None.
Remarks:
Doses / Concentrations:
1250, 2500 or 5000 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
8
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
- Route of administration: oral gavage
- Doses / concentrations: 64 mg/kg
Tissues and cell types examined:
After sacrifice the bone marrow from the shafts of both femurs was removed and smears were prepared on slides.

Details of tissue and slide preparation:
Routinely the slides of five animals/sex/dose were stained with May-Grunwald solution and scored for micronuclei. As an exception, the slides from 3 males and 7 females which were killed after 16 hours (part 1) were scored, since only three males in each group furnished well differentiated cells.
Evaluation criteria:
From each animal the ratio of polychromatic to normochromatic erythrocytes was determined as an indicator for toxic effects on the erythropoiesis and 1000 polychromatic erythrocytes were scored for micronuclei.
Statistics:
Chi-Square test used
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In both parts of the main experiment all hamsters survived. At all sampling times (16, 24, and 48 hours) and at all dose levels (5000, 2500, and 1250 mg/kg) no statistically significant increase of micronucleated polychromatic erythrocytes (PCEs) was observed when compared to the negative control. In the positive control groups the percentage of micronucleated PCEs was statistically significant increased, thus demonstrating the sensitivity of the test system.

Chinese hamster micronucleus test

Part 1: Percentage of micronucleated PCEs at different preparation times

Test article and concentration

Sex

16 hours

24 hours

48 hours

CGA154281

(5000 mg/kg)

males

females

mean

0.07

0.06

0.06

0.00

0.08

0.04

0.06

0.06

0.06

Negative control – vehicle

(0.5% CMC)

males

females

mean

0.07

0.04

0.05

0.04

0.04

0.04

0.10

0.04

0.07

Positive control – cyclophoshamide

(64 mg/kg)

males

females

mean

 

3.68

5.46

4.57*

 

* p>0.05 (Chi-Square test)

Chinese hamster micronucleus test

Part 2: Percentage of micronucleated PCEs at different dose levels sacrificed after 24 hours

Sex

CGA154281

1250 mg/kg

CGA154281

2500 mg/kg

CGA154281

5000 mg/kg

Negative control

0.5% CMC

Positive control

64 mg/kg CPA

Males

Females

Mean

0.02

0.00

0.01

0.04

0.02

0.03

0.04

0.06

0.05

0.08

0.02

0.05

2.00

1.58

1.79*

* p > 0.05 (Chi-Square Test)

Conclusions:
Interpretation of results (migrated information): negative
Under the given experimental conditions no evidence for a clastogenic or aneugenic activity of CGA154281 was observed in vivo.
Executive summary:

CGA154281 technical, Benoxacor, was administered by gavage to Chinese hamsters to evaluate any mutagenic activity on polychromatic erythrocytes in bone marrow cells, in vivo. In the first experiment the highest dose of 5000 mg/kg was administered and animals sacrificed 16, 24 or 48 hours later. In the second assay, doses of 1250, 2500 and 5000 mg/kg were administered and animals sacrificed after 24 hours exposure.

In the first assay, bone marrow smears showed no significant increase in micronucleated polychromatic erythrocytes.

In the second assay, bone marrow smears showed no significant increase in micronucleated polychromatic erythrocytes.

No evidence of mutagenic effects were found in the Chinese hamster after oral administration of Benoxacor.

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

Additional information

Additional information from genetic toxicity in vivo:

In vitro data

The key studies are considered to be two bacterial mutation assays (Ogorek, 1987a and b), a mammalian cell gene mutation assay (Dollenmeier, 1987) and a DNA repair assay (Hertner, 1987a). These are recognised core assay types for investigating mutation in vitro.

The test material has been tested in several bacterial mutation assays, which measure the induction of point and frame shift mutations in strains of Salmonella typhimurium with and without auxiliary metabolic activation (S9). The number of studies reflects the testing of different development batches. The key studies selected represent material currently manufactured on a production scale (of purity 95.4 to 98.3%; Ogorek, 1987a and b) and demonstrate that benoxacor is clearly negative at concentrations up to 5000 µg/plate. Furthermore, highly purified material (purity 99.3%) was also negative in this assay (Deparade, 1984). A weak mutagenic response was observed with an early batch of lower purity (93.9%; Deparade, 1986; Lasinski at al, 1986), however, a weight of evidence evaluation, and in particular including batches of purity relevant to production material, indicates that benoxacor can be concluded to be negative in the bacterial mutation assay.

The bacterial mutation assays which tested a range of purities are summarised below.

Gene mutation in vitro (Ames assays), listed in order of sample purity, with the key studies shown in bold:

Strains tested

Concentration tested

Batch &

purity of CGA154281

Result

Reference

non-activated

activated

4 strains:

TA 98, TA100, TA1535, TA1537

20, 80, 320, 1280, 5120 ug/0.1 mL

HM4287/1+4

99.3%

Negative

Negative

Deparade, (1984)

4 strains:

TA 98, TA100, TA1535, TA1537

20, 78, 313, 1250 and 5000 µg/0.1 mL

FL870116

98.3%

Negative

Negative

Ogorek, (1987a)

4 strains:

TA 98, TA100, TA1537, TA1538

313, 625, 1250, 2500, 5000 ug/0.1 mL

FL881310

97.1%

Negative

Negative

Ogorek, (1988)

3 strains:

TA 98, TA1537, TA1538

250, 500, 1000, 2000, 3000, 4000, 5000 and 8000 µg/0.1 mL

FL870221

95.4%

Negative

Negative

Deparade, (1988a)

3 strains:

TA 98, TA1535, TA1537

250, 500, 1000, 2000, 3000, 4000, 5000 and 8000 µg/0.1 mL

FL870221

95.4%

Negative

Negative

Deparade, (1988b)

4 strains:

TA 98, TA100, TA1535, TA1537

20, 78, 313, 1250 and 5000 µg/0.1 mL

FL870221

95.4%

Negative

Negative

Ogorek, (1987b)

5 strains:

TA98, TA100, TA102, TA1535 and TA1537

20, 78, 313, 1250 and 5000 µg/0.1 mL (all strains) and 500, 1000, 2000, 4000 and 8000 µg/0.1 mL (strain TA98)

P.510001

93.9%

Weak response in TA98 at 2000 µg/0.1 mL and above seen in only some experiments

Weak response in TA98 at 4000 µg/0.1 mL and above seen in only some experiments

Deparade, (1986)

5 strains:

TA98, TA100, TA1535, TA1537 and TA1538

5, 10, 50, 250, 1000 µg/plate

FL860318

93.9%

Weak response in TA98, TA1537 and TA1538 at 1000 µg/plate

Weak response in TA98, TA1537 and TA1538 at 1000 µg/plate

Lasinski

 

The test material was investigated for the induction of point mutation in a mammalian cell mutation assay in Chinese hamster V79 cells in vitro. The test material was examined at concentrations up to 120 µg/mL and no mutagenic effects were observed with or without metabolic activation (Dollenmeier, 1987). This finding supports the conclusion from the bacterial mutation assays, that benoxacor is negative for the gene mutation endpoint.

Benoxacor has been examined in a DNA-repair assay using material typical of current manufacture and does not induce DNA-damage in rat hepatocytes at concentrations up to 20 µg/mL, a dose limited by the toxicity of the test material. This key assay is reported by Hertner, 1987a. Furthermore, no DNA-damage was induced in a DNA-repair test on human fibroblasts tested at concentrations up to 62.5 µg/mL (Meyer, 1986), or in rat hepatocytes and human fibroblasts tested with highly purified material (Puri, 1985a and b). Evidence for a possible weak induction of DNA-repair was reported in rat hepatocytes with an early developmental batch of lower purity (Puri, 1986), however a negative result was reported for the same purity of material in human fibroblasts (Meyer, 1986). A weight of evidence evaluation, and in particular including batches of purity relevant to production material, indicates that benoxacor is negative in the DNA repair assay in vitro.

Autoradiographic DNA repair assays, listed in order of sample purity, with the key study shown in bold:

Test system

Concentration tested

Batch &

purity of CGA154281

Result

Reference

Human fibroblasts

0.25, 1.25, 6.25, 31.25 ug/ml

KGL 3339-6

99.7%

No effects on DNA

Puri, (1985a)

Rat hepatocytes

1,5, 25, 125ug/ml

Not reported

99.7%

No effects on DNA

Puri, (1985b)

Rat hepatocytes

original study: 0.008 to 20 ug/ml confirmatory study: 0.0004 to 10 ug/ml

FL870116

98.3%

No effects on DNA

Hertner, (1987b)

Rat hepatocytes

original study: 0.3 to 30 ug/ml confirmatory study: 0.1 to 20 ug/ml

FL870211 95.4%

No effects on DNA

Hertner, (1987a)

Human fibroblasts

0.5, 2.5, 12.5, 62.5 ug/ml

P. 510001  93.9%

No effects on DNA

Meyer, (1986)

Rat hepatocytes

original study: 0.12 to15 ug/ml confirmatory study: 0.125 to 20 ug/ml

P. 510001 Purity 93.9%

Marginal induction of DNA damage

Puri, (1986)

 

In vivo data

The key study in vivo is a bone marrow micronucleus assay in the Chinese hamster (Stasser, 1986). This assay tests for clastogenic and aneugenic activity and is a recognised core assay type for investigating mutation in vivo. The test material (93.9% purity) was tested at dose levels up to 5000 mg/kg b.w and samples of bone marrow cells were taken for cytogenetic analysis at 16, 24 and 48 hours after oral dosing. No significant increases in micronucleated polychromatic erythrocytes were found. Benoxacor is negative in this assay for clastogenic and aneugenic activity.

Summary

The test material has been examined for mutagenicity both in vitro and in vivo in a range of recognised core assay types. All in vitro studies conducted with material representative of currently manufactured technical material or material of higher purity were negative. Weak genotoxic responses in vitro were only obtained with early developmental material of lower purity levels. A sample of such lower purity material was tested for mutagenicity in vivo and gave a negative result. It is concluded that the available data indicate that benoxacor has no significant genotoxicity.

 

 


Justification for selection of genetic toxicity endpoint
One reliable, GLP- and guideline-conform in vivo genotoxicity study was available demonstrating that the substance is not mutagenic. This finding is supported by a set of available reliable and valid in vitro genotoxicity assays focussing on different endpoints of genotoxicity.

Justification for classification or non-classification

The available data on genotoxicity are adequate for the risk assessment, classification and labelling. The mutagenic potential of benoxacor has been assessed in a number of core genotoxicity assays in vivo and in vitro, covering a range of endpoints including gene mutation, DNA repair and clastogenicity. The data indicate the test material is not genotoxic.

Based on the results, the substance does not meet the criteria for classification under the Directive 2001/59/EC, Annex VI, 4.2.2 as well as the criteria in the Regulation (EC) No. 1272/2008, Annex I, Part 3, 3.5, as amended by Regulation (EC) No. 286/2011.