N'-(1,3-dimethylbutylidene)-3-hydroxy-2-naphthohydrazide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

BMH was tested in three in vitro studies

Bacterial reverse mutation assay (Hita K01 -2089) conducted similar to the OECD 471 test guideline: Negative in TA1535, TA1537, TA100 and TA98 and WP2uvrA in absence and presence of metabolic activation;

Bacterial reverse mutation assay (Notox 300634) conducted according to the OECD 471 test guideline: Positive in S. typhimurium TA 100 in absence of metabolic activation;

In vitro Mouse Lymphoma assay (Notox 318083) conducted according to the OECD 476 test guideline: Negative in presence and absence of metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 July 1998 - 14 July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

according to guideline

Guideline:

other: see "Principles of method if other than guideline"

Version / remarks:

Similair to OECD TG 471

Deviations:

yes

Remarks:

see "Principles of method if other than guideline"

Principles of method if other than guideline:

- According to the method described in "Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances' (Notification No. 700 of the Planning and Coordination Bureau, EA, No. 1039 of the Pharmaceutical Affairs Bureau, MHW & No. 1014 (1986) of the Basic Industries Bureau, MITI, December 5, 1986 and Notification No. 287 of the Planning and Coordination Bureau, EA, No. 127 of the Environmental Health Bureau, MHW & No. 2 (1997) ofthe Basic Industries Bureau, MITI, October 31, 1997).

- Deviations: No justification for the use of duplicate test cultures instead of triplicate test cultures

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 induced with phenobarbital and 5,6-benzoflavone
- Composition of S9 mix: S9-mix per mL: 4 μmol NADPH, 4 μmol NADH 5 μmol glucose-6-phosphate; 100 μmol sodium phosphate buffer pH 7.4; 8 μmol MgCl2; 33 μmol KCl and 0.1 mL of S9

Test concentrations with justification for top dose:

Dose range finding test:
S. typhimurium TA100, TA98, TA1535, TA1537 and E. coli WP2 uvr A: 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate in the absence and presence of S9-mix.

Main Experiment (without S9-mix):
S. typhimurium TA100 and TA1535: 19.5, 39.1, 78.1, 156, 313 and 625 µg/plate
S. typhimurium TA98, TA1537 and E. coli WP2 uvr A: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

Main Experiment (with S9-mix):
S. typhimurium TA100,TA1535 TA98, TA1537 and E. coli WP2 uvr A: 39.1, 78.1, 156, 313, 625 and 1250 µg/plate

Justification for Top dose: Limit of cytotoxicity

Vehicle / solvent:

- Vehicle used: DMSO
- The Substance was stable and dissolved in DMSO (no change in the 50 mg/mL solution prepared with dehydrated DMSO, including such as color change and heat generation, until 2 hours after preparation was observed)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: AF-2 (2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide)

Remarks:

In DMSO; Without S9: TA100 (0.01 µg/plate); WP2 uvr A (0.01 µg/plate); TA98 (0.1 µg/plate)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

In distilled water; Without S9: TA1535 (0.5 µg/plate)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: ICR-191 (2-Methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine.2HCl)

Remarks:

In DMSO; Without S9: TA1537 (1 µg/plate)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2AA (2-Aminoanthracene)

Remarks:

In DMSO: With S9: TA100 (1 µg/plate), TA1535 (2 µg/plate), TA98 (0.5 µg/plate), TA1537 (2 µg/plate) and E. coli WP2 uvr A (10 µg/plate)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Positive control and test substance concentrations: duplicate
Negative control: Triplicate

- Number of independent experiments: 2 (dose range finder and main experiment)

METHOD OF TREATMENT/ EXPOSURE:
In agar (plate incorporation); preincubation


TREATMENT AND HARVEST SCHEDULE
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

COLONY COUNTING
The number of revertant colonies was counted using the manual counter or the colony analyzer (CA-7, Toyo-sokki Co., Ltd). With the colony analyzer, the count was finalized after correcting for area and count drop. Each plate was measured twice, and the average of these two measurements was adopted as the number of revertant colonies on the plate.

NUMBER OF CELLS EVALUATED: > 2E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: background growth inhibition
- Other: precipitation of the test item was recorded by visual inspection.

Evaluation criteria:

The test substance is considered positive when the number of revertant colonies increased twice or more that of the negative control in a dose-dependent manner given the results are reproducible. When these conditions are not met the substance is considered negative.

Statistics:

Not applicable.

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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

RANGE-FINDING/SCREENING STUDIES:
Precipitation of the test item on the plates was observed at concentrations of 1250 µg/plate and higher. In the dose-range finding study growth was inhibited at 313 µg/plate and above in TA100 and TA1535 and at 1,250 µg/plate and above in WP2 uvrA, TA98 and TA 1537 in the absence of S9 mix. Growth was inhibited in all tester strains at 1250 µg/plate and higher in the presence of S9 mix.
Based on the results of the dose range finding test, 625 µg/plate of strains of TA100 and TA1535, and 1,250 µg/plate for strains of WP2 uvrA, TA98 and TA1537 in the absence of S9 mix, and 1,250 µg/plate for all of the tester strains in the presence of S9 mix were determined as the highest dose for the main test.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive controls induced significant increase in the number of revertant colonies and the number of revertant colonies in the positive controls and the negative control was within the range of the historical laboratory background data. These results confirm that the test conditions were adequate and that the metabolic activation system functioned properly.
Ames test:
- Signs of toxicity : In the main study growth was inhibited at 313 µg/plate for TA100 and TA1535, at 625 µg/plate for TA98 and TA1537 and 1250 µg/plate for WP2 uvrA in the absence of S9. Growth inhibition was observed at 1,250 ug/plate in all of the test strains in the presence of S9 mix.
- Mean number of revertant colonies per plate and standard deviation : The number of revertant colonies for all the test strains was less than twice that of the negative control with and without S9 mix. (see attached background material for detailed results)

Conclusions:

Based on the results of an Ames test performed according to a guideline similair to OECD 471 and in accordance with GLP principles, BMH is concluded to be not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

A bacterial reverse mutation test was performed according to the method described in : Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances"; similair to OECD 471 and in accordance with GLP principles. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate. Cell growth was inhibited at 313 µg/plate and above in TA100 and TA1535 and at 1,250 µg/plate and above in WP2 uvrA and at 625 µg/plate and above in TA98 and TA 1537 in the absence of S9 mix. Growth was inhibited in all tester strains at 1250 µg/plate and higher in the presence of S9 mix. Precipitation of the test item on the plates was observed at concentrations of 1250 µg/plate and higher. Based on the results of the dose range finding test, 625 µg/plate for strains of TA100 and TA1535, and 1,250 µg/plate for strains of WP2 uvrA, TA98 and TA1537 in the absence of S9 mix, and 1,250 µg/plate for all of the tester strains in the presence of S9 mix were determined as the highest doses for the main test. In the main study growth was inhibited at 313 µg/plate for TA100 and TA1535,  at 625 µg/plate for TA98 and TA1537 and 1250 µg/plate for WP2 uvrA in the absence of S9. Growth inhibition was observed at 1,250 ug/plate in all of the test strains in the presence of S9 mix. Positive and negative controls were included and showed that the test system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant colonies in the tester strains (TA1535, TA1537, TA98 and TA100 and WP₂uvrA) both in the absence and presence of S9 -metabolic activation when tested up to the limit of cytotoxicity. Based on the results of this study it is concluded that BMH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay. 

Reference 2

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:

08 November 2000 - 08 January 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Dose range finding test:
S. typhimurium TA100 and E. coli WP2 uvr A: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix. This dose range finding test was reported as part of the 1st experiment of the main study.
Experiment 1:
S. typhimurium TA98, TA1535 and TA1537: 33, 100, 167, 333, 666 and 1000 µg/plate in the absence and presence of 5% (v/v) S9-mix.
Experiment 2:
S. typhimurium TA98, TA100 and TA1537: 33, 100, 167, 333 and 666 µg/plate in the absence and presence of 10% (v/v) S9-mix.
S. typhimurium TA1535: 10, 33, 100, 333 and 1000 µg/plate in the absence and presence of 10% (v/v) S9-mix.
E. coli WP2 uvr A: 33, 100, 333, 1000 and 2000 µg/plate in the absence of S9-mix.
E. coli WP2 uvr A: 33, 100, 333, 1000 and 3330 µg/plate in the absence and presence of 10% (v/v) S9-mix.
Experiment 3:
S. typhimurium TA100: 100, 150, 200, 250, 300 and 350 µg/plate in the absence and presence of 5% (v/v) S9-mix.

Vehicle / solvent:

Solvent: Dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Migrated to IUCLID6: 60 µg/plate in saline for TA1537

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Daunomycine, 4 µg/plate in saline for TA98

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Not performed.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

333 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1000 µg/plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA1535 and TA1537

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 666 and 1000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 1000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98, TA100 and TA5137

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 666 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 2000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

( 350 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
The negative and strain-specific positive control values
were within our laboratory background historical control
data ranges indicating that the test conditions were
adequate and that the metabolic activation system functioned
properly.


In tester strain TA100 in the absence of S9-mix, the test
substance induced up to 3.2- and 2.4-fold increases in the
number of revertant colonies compared to the solvent
controls in the first and second experiment respectively.
Verification of this result was performed in the additional
third experiment, in which the test substance showed a 1.8-
fold, dose-related, increase in the number of revertant
colonies compared to the solvent control. The increases
(more than two-fold) were observed in two out of three
experiments and a dose related increase (just below two-
fold) was observed in one experiment. Furthermore, the
increases were outside our historical control data range.
Therefore, these increases were considered to be
biologically relevant and the test substance is
considered to be mutagenic in tester strain TA100 in the
absence of S9-mix.

In the presence of 5% (v/v) S9-mix, the test substance
induced an up to 2.1-fold increase in the number of
revertant colonies compared to the solvent control in the
first experiment. No two-fold increase in the number of
revertant colonies was observed after the modification in
the composition of the S9-mix (10% (v/v) S9 fraction) in the
second experiment. Verification of the result of the first
experiment was performed in the additional third experiment
with 5% (v/v) S9-fraction, in which the test substance
showed no two-fold increase in the number of revertant
colonies. Therefore, the increase was considered to be not
biologically relevant and the test substance is considered
to be not mutagenic in tester strain TA100 in the presence
of S9-mix.

In the other four tester strains (TA1535, TA1537, TA98 and
WP2uvrA) in the absence and presence of S9-mix, BMH did not
induce a dose-related increase in the number of revertant
colonies in independently repeated experiments.

Remarks on result:

other: other: preliminary test

Remarks:

Migrated from field 'Test system'.

Summary Tables of the mutagenic response of BMH are attached in the background material.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation in strains TA1535, TA1537, TA98 and WP2uvrA
positive without metabolic activation in strain TA100 only

BMH was tested in the Salmonella typhimurium reverse mutation assay with TA1535, TA1537, TA100 and TA98 and in the Escherichia coli reverse mutation assay with Escherichia coli WP2uvrA according to OECD Guideline 471 and GLP principles.
BMH showed a dose related increase in the number of revertant colonies in strain TA100 in the absence of S9-mix.
In the other four tester strains (TA1535, TA1537, TA98 and WP2uvrA) in the absence and presence of S9-mix, and in strain TA100 in the presence of S9-mix, BMH did not induce a dose-related increase in the number of revertant colonies in independently repeated experiments.

Reference 3

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:

January 16 - February 27, 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and penicillin/streptomycin (50 U/ml and 50 μg/ml respectively).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate of rat treated with Aroclor 1254.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 0, 1, 10, 33 and 100 µg/ml
Without S9-mix, 24 hours treatment: 0, 1, 10, 33 and 100 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0, 1, 5, 10, 20, 25, 30, 35, 40, 50, 60 and 70 µg/ml
With S9-mix, 3 hours treatment: 0, 0.1, 0.25, 0.5, 1, 2.5, 5, 7.5, 10, 15 and 20 μg/ml
Experiment 2:
Without S9-mix, 24 hours treatment: 0, 5, 10, 20, 25, 30, 40, 50, 60, 70, 75 and 80 μg/ml
With S9-mix, 3 hours treatment: 0, 0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25 and 30 μg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Migrated to IUCLID6: final concentration of 2 mM for a 3 h treatment period and 1 mM for a 24 h treatment period

Negative solvent / vehicle controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

other: Dimethylnitrosamine, final concentration of 0.5 mM

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 10 to 11 days

SELECTION AGENT (mutation assays): 5 µg/mL 5-trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Single, solvent controle: duplicate

NUMBER OF CELLS EVALUATED: total number of 9.6 x 10^5 cells

DETERMINATION OF CYTOTOXICITY
- Method: suspension growth (dose range finding test) and cloning efficiency (experiment 1 and 2)

Evaluation criteria:

A test substance was considered positive (mutagenic) in the mutation assay if:
a) It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and
b) The results were reproducible in an independently repeated test.

A test substance was considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
b) The results were confirmed in an independently repeated test.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

Not performed.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(> 10 µg/ml)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Dose range finding test: the substance precipitated in the exposure medium at a concentration of 100 μg/ml.
Experiment 1 and 2: a concentration of 50 µg/ml BMH already precipitated in the exposure medium.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix after 3 hours of treatment the toxicity in the suspension growth was 54% at the test substance concentration of 33 μg/ml compared to the suspension growth of the solvent control. No cell survival was observed at the test substance concentration of 100 μg/ml after 3 hours treatment.
In the presence of S9-mix after 3 hours of treatment, the toxicity in the suspension growth was 92% at the test substance concentration of 10 μg/ml compared to the suspension growth of the solvent control. No cell survival was observed at test substance concentration of 33 and 100 μg/ml after 24 hours subculture.
In the absence of S9-mix after 24 hours of treatment, the toxicity in the suspension growth was 62% at the test substance concentration of 33 μg/ml compared to the suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 100 μg/ml after 24 hours subculture.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutant frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Mutant frequencies in cultures treated with positive control chemicals were increased by 9.3- and 30-fold for EMS in the first and second experiment respectively, and by 14- and 15-fold for DMN, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly.

Remarks on result:

other: strain/cell type: L5178Y mouse lymphoma cells

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The mouse lymphoma assay was conducted according to OECD 476 and GLP guidelines.

In the absence of S9-mix, BMH induced a 9.8-fold, dose-related, increase in the mutant frequency at the TK locus in the second experiment. However, this increase was observed only at very toxic (>94%) and precipitating concentrations (70 and 80 μg/ml). Furthermore, this increase was observed after a prolonged treatment time only. Therefore, this increase was considered to be not biologically relevant and BMH is considered to be not mutagenic in the absence of S9-mix.

In the presence of S9-mix, BMH induced no significant increase in the mutant frequency in both independent experiments. The modification in the composition of the S9-mix showed no amplification of the mutagenic effect at the TK-locus.

In conclusion, BMH is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus study in mouse (Notox 367864) conducted according to the OECD 474 test guideline: Negative (not clastogenic)

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:

December 03, 2002 - January 22, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: NMRI BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: Males: 30.6-32.8 g, females: 24.2-25.8 g
- Housing: Group housing of 5 animals per sex per cage in labelled polycarbonate cages containing purified sawdust as bedding material (Sawi, Jelu Werk, Rosenberg, Germany). Paper bedding was provided as nest material (supplied by B.M.I. Helmond, The Netherlands).
- Diet (e.g. ad libitum): Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany).
- Water (e.g. ad libitum): Free access to tap-water.
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

Results of analysis for diet (nutrients and contaminants) and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
BMH was suspended in corn oil (Roth, Karlsruhe, Germany). BMH concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. BMH concentrations were dosed within 4 hours after preparation.

The dosing volume was 10 ml/kg body weight.

The route and frequency of administration and the volume administered of the negative and the positive control was the same as those of the test article.

Duration of treatment / exposure:

Dose range finding study: One dose group, comprising 3 males and 3 females, received a single dose of BMH.
The study duration was one to three days.

Micronucleus test:
Test substance groups: 24 and 48 hours; Vehicle group: 24 hours; Positive control group: 48 hours

Frequency of treatment:

Once

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: single intraperitoneal injection
- Doses / concentrations: 50 mg/kg body weight (10 ml/kg bw)

Tissues and cell types examined:

Measuring the increase of the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in mouse bone marrow.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the Micronucleus test was based on a dose range finding study. Results see table 1 in section "Any other information on results incl. tables".

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The animals were sacrificed by cervical dislocation 24 or 48 h (2000 mg/kg only) after dosing of BMH, 24 h after dosing of the vehicle and 48 h after dosing of the positive control.

DETAILS OF SLIDE PREPARATION:
Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes neither in the combined data for both sexes nor in the data for male or female groups separately.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

Wilcoxon Rank Sum Test; two-sided test.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Doses producing toxicity: In the range finding no animals died up to the highest dose (2000 mg/kg bw). In the micronucleus test, two female animals from the 24 h sampling group (B) died within 18 to 24 hours after dosing.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
The animals of the negative and positive control groups showed no abnormalities.

The following clinical observations were made in the groups treated with 2000 mg/kg body weight BMH:

During the first hour after dosing all animals showed no reaction to treatment.

Within 18 hours after dosing one male animal showed no reaction to treatment, the other nine male animals had a hunched posture, one animal was also lethargic and one animal also had a rough coat. Within 18 hours after dosing all female animals were lethargic and had a hunched posture, eight female animals also showed ventro-lateral recumbency and five out of these eight animals also had ptosis. Within 18 to 24 h after dosing two female animals died.

Within 42 hours after dosing one male animal still showed no reaction to treatment, the other four male animals had a hunched posture and a rough coat and one animal was also lethargic. All female animals had recovered from the treatment.

The following clinical observations were made in the group treated with 1000 mg/kg body weight:

During the first hour after dosing all animals showed no reaction to treatment.

Within 18 hours after dosing all male animals and one female animal had a hunched posture, two male animals were also lethargic. The other four females animals showed no reaction to treatment.

One male animal treated with 500 mg/kg body weight had a rough coat the first hour after dosing.

Within 18 hours after dosing the animal recovered from the treatment. All other animals treated with 500 mg/kg body weight showed no abnormalities after dosing.

TABLE 1 MORTALITY AND SYSTEMIC TOXIC SIGNS AFTER TREATMENT WITH BMH IN THE DOSE RANGE FINDING STUDY

Group          Sex                     Animal           Dose                                  Systemic toxic signs*

                                          number          mg/kg                        day 1 within                      day 2              day 3

                                                                                            

30 min.    1 hr.    2 hrs.

 

A                   Male                     1                 2000                B             B                  X                   X

A                   Male                     2                 2000                B                       F         B                    B

A                   Male                     3                 2000                B                       CF       NX                  FNX

A                   Female                  4                 2000                B            CF               FNX                 X

A                   Female                  5                 2000                B                        CF      FXY                 FNX

A                   Female                  6                 2000                B                        CF      FXY                 B

 

* Legend 'Mortality and systemic toxic signs':

B = showed no abnormalities; C = ataxia; F = lethargy; N = rough coat; X = hunched posture; Y= ventro-lateral recumbency

Micronucleus test:
A vehicle treated group served as negative control, a group treated with a single intraperitoneal injection of cyclophosphamide (CP) at 50 mg/kg body weight served as positive control.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of
micronucleated polychromatic erythrocytes in both sexes.

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes
of the bone marrow of animals treated with the test substance.

The groups that were treated with the test substance showed no decrease in the ratio of polychromatic to normochromatic
erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the
erythropoiesis. The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls.

Conclusions:

Interpretation of results (migrated information): negative
An in vivo micronucleus study with BMH in the mouse (5 animals per sex/dose, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. As the route of administration was an intraperitoneal injection and toxicity was observed, it is considered that the test substance has reached the target tissue. It is concluded that BMH is not clastogenic in the micronucleus test under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

A bacterial reverse mutation test was performed according to the method described in :Notification on Partial Revision of Testing Methods Relating to the New Chemical Substances";similair to OECD 471 and in accordance with GLP principles. In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate. Cell growth was inhibited at 313 µg/plate and above in TA100 and TA1535 and at 1,250 µg/plate and above in WP2 uvrA and at 625 µg/plate and above in TA98 and TA 1537 in the absence of S9 mix. Growth was inhibited in all tester strains at 1250 µg/plate and higher in the presence of S9 mix. Precipitation of the test item on the plates was observed at concentrations of 1250 µg/plate and higher. Based on the results of the dose range finding test, 625 µg/plate for strains of TA100 and TA1535, and 1,250 µg/plate for strains of WP2 uvrA, TA98 and TA1537 in the absence of S9 mix, and 1,250 µg/plate for all of the tester strains in the presence of S9 mix were determined as the highest doses for the main test. In the main study growth was inhibited at 313 µg/plate for TA100 and TA1535, at 625 µg/plate for TA98 and TA1537 and 1250 µg/plate for WP2 uvrA in the absence of S9. Growth inhibition was observed at 1,250 ug/plate in all of the test strains in the presence of S9 mix. Positive and negative controls were included and showed that the test system functioned properly. The test item did not induce a significant dose-related increase in the number of revertant colonies in the tester strains (TA1535, TA1537, TA98 and TA100 and WP₂uvrA) both in the absence and presence of S9 -metabolic activation when tested up to the limit of cytotoxicity. Based on the results of this study it is concluded that BMH is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia colireverse mutation assay. 

In a second Ames test BMH was tested with TA1535, TA1537, TA100, TA98 and Escherichia coli WP2uvrA according to OECD Guideline 471 and GLP principles. BMH showed a dose related increase (more than two-fold), in two out of three experiments, in the number of revertant colonies in strain TA100 in the absence of S9-mix and is considered to be mutagenic in this strain. In the other four tester strains (TA1535, TA1537, TA98 and WP2uvrA) in the absence and presence of S9-mix, and in strain TA100 in the presence of S9-mix, BMH did not induce a dose-related increase in the number of revertant colonies in independently repeated experiments.

In the mouse lymphoma assay in the absence of S9-mix, BMH induced a 9.8-fold, dose-related, increase in the mutant frequency at the TK locus in the second experiment. However, this increase was observed only at very toxic (>94%) and precipitating concentrations (70 and 80 μg/ml). Furthermore, this increase was observed after a prolonged treatment time only. Therefore, this increase was considered to be not biologically relevant and BMH is considered to be not mutagenic in the absence of S9-mix. In the presence of S9-mix, BMH induced no significant increase in the mutant frequency in both independent experiments. The modification in the composition of the S9-mix showed no amplification of the mutagenic effect at the TK-locus. In conclusion, BMH is not mutagenic in the TK mutation test system under the experimental conditions.

An in vivo micronucleus study with BMH in the mouse (5 animals per sex/dose, intraperitoneal injection) was conducted according to OECD 474 and GLP guidelines. As the route of administration was an intraperitoneal injection and toxicity was observed, it is considered that the test substance has reached the target tissue. It is concluded that BMH is not clastogenic in the micronucleus test under the experimental conditions described in this report.

Justification for classification or non-classification

No classification can be considered for mutagenicity as positive in vitro data indicates that further information is needed to conclude on the ability of BMH to induce gene mutations