1,1,4,4-tetramethylbutane-1,4-diyl bis(2-ethylperoxyhexanoate)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Justification for selection of genetic toxicity endpoint

Ames + MLA + in vivo micronucleus, K1: The study was performed according to OECD guidelines and GLP.

Short description of key information:

The substance is positive for mutagenicity with metabolic activation in vitro in an Ames test and negative in a Mouse Lymphoma Assay.

In accordance with Endpoint Specific Guidance Chapter R.7A, Figure R.7.7-1 "Flow chart of the mutagenicity testing strategy", no further testing (i.e. no in vivo testing) need be proposed in the event of a negative mouse lymphoma assay or hprt assay, regardless to whether or not the gene mutation test in bacteria is positive or negative.  This therefore implies that when considering whether an in vivo gene mutation request is required for substances requiring Annex IX test proposals due to their volume bands, a negative mouse lymphoma assay or hprt assay is sufficient evidence to waive the need for an in vivo gene mutation test.

There is no in vitro cytogenicity study available however an in vivo micronucleus study is negative.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

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:

23 December 1999 - 10 March 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD guidelines and GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see below

Species / strain / cell type:

S. typhimurium TA 102

Additional strain / cell type characteristics:

other: see below

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

preliminary tes, with and withou S9-mixt: 10, 100, 500, 1000, 2500 and 5000 µg/plate.

Experiments without S9 mix:
The selected treatment-levels were:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains in both mutagenicity experiments, except for the TA 98 strain in the second experiment, 625, 1250, 2500, 3750 and 5000 µg/plate, for the TA 98 strain in the second experiment.

Experiments with S9 mix:
The selected treatment-levels were:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all tester strains In both mutagenicity experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility
Batch No. 1743850 731 (Merck Clevenot, 77500 Chelles, France).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: see below

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 60 min.
- Exposure duration: 48/72 hours
- Expression time (cells in growth medium): 48/72 hours

SELECTION AGENT (mutation assays): histidine

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: revertants were scored with an automatic counter (Anek counter, model 880, OS1, 750] 5 Paris, France).

DETERMINATION OF CYTOTOXICITY: The evaluatiDn of the tDxicity was perfonned Dn the basis of the observation Df the decrease in the number of revertant cDlonies andlDr a thinning of the bacterial lawn.

Evaluation criteria:

Acceptance criteria
This study would be considered valid since the following criteria are fully met:
· the number of revertants in the vehicle controls is consistent with our historical data,
· the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with our historical data.

Evaluation criteria
A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

None

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
The test substance was soluble in the vehicle (DMSO) at 50 mg/ml. With a maximum dose volume of 100 µg/plate. No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. No toxicity was noted towards the three strains used, with and without S9 mix.

In the second experiment (preincubation method), at dose-levels ≥ 625 µg/plate, a 1.8-2.4 fold and a 2-3.7 fold increase in the number of revertants were noted in the TA 98 and TA 100 strain, respectively.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on TA 98 and TA 100 Salmonella typhimurium strains, with metabolic activation.

Executive summary:

A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study. The test substance was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except the second with S9 mix, which was performed according to the preincubation method (60 minutes, 37C).

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA102 were used. Each strain was exposed to five dose-levels of the test substance (three plates/dose-level).

After 48 to 72 hours of incubation at 37C, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn. The test substance was dissolved in dimethylsulfoxide (DMSO).

The test substance was soluble in the vehicle at 50 mg/ml. Consequently, with a maximum dose volume of 100 µg/plate, the dose-levels for the preliminary toxicity test were: 10, 100, 500, 1000, 2500 and 5000 µg/plate. Since the test substance was non-toxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

Experiments without S9 mix:

The selected treatment-levels were:

312.5. 625, 1250. 2500 and 5000 µg/plate for all tester strains in both mutagenicity experiments except for the TA 98 strain in the second experiment. 625, 1250.2500, 3750 and 5000 µg/plate for the TA 98 strain in the second experiment. A slight to moderate emulsion was hoted in the plates at dose levels ≥ 1250 µg/plate. No or a slight toxicity was noted depending on the tester strain and the dose-level. No mutagenic effect was noted in all the tester strains.

Experiments with S9 mix:

The selected treatment-levels were:

312.5. 625. 1250. 2500 and 5000 µg/plate for all tester strains in both mutagenicity experiments.

A slight to moderate emulsion was generally noted in the plates at dose-levels ≥1250 µg/plate. No or a slight to moderate toxicity was noted. depending on the tester strain, the dose-levels and experimental conditions.

In the second experiment (preincubation method), at dose-levels ≥ 625 µg/plate, a 1.8-2.4 fold and a 2-3.7 fold increase in the number of revertants were noted in the TA 98 and TA 100 strain, respectively.

The number of revertants of the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

Under our experimental conditions, the test substance induced mutagenic activity in the bacterial reverse mutation test on TA 98 and TA 100

Salmonella typhimurium strains, with metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

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:

05 September 2011 and 24 October 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

According to OECD 476. Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

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

Principles of method if other than guideline:

first experiment: 4 hours treatment with and without metabolic activation
second experiment: 24 hours treatment without metabolic activation, 4 hours treatment with metaoblic activation

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

Experiment I:
without S9 mix (4 hours treatment): 8.8; 17.5; 35.0; 70.0; 140.0; 210.0; and 280.0 µg/mL
with S9 mix (4 hours treatment): 70.0; 140.0; 280.0; 560.0; 840.0; and 1120.0 µg/mL
Experiment II:
without S9 mix (24 hours treatment): 2.2; 4.4; 8.8; 17.5; 35.0; 52.5; and 70.0 µg/mL
with S9 mix (4 hours treatment): 35.0; 70.0; 140.0; 280.0; 560.0; and 840.0 µg/mL
On the day of the experiment (immediately before treatment), the test item was dissolved in acetone. The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. The final concentration of acetone in the culture medium was 0.5 % v/v. The complete dilution series in acetone was kept on ice until application to the cell cultures.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours with and without metabolic activation in experiment 1, 24 hours without metaoblic activation in experiment and 4 hours with metabolic activation in experiment 2
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1,5 x 10 exp. 6 cells

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 10 exp. 6 cells above the
corresponding solvent control or negative control, respectively.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in negative
and/or vehicle con¬trols and the mutation rates of all negative and/or vehicle controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth, and the cloning efficiency 1 is less than 10 % of the vehicle control
unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used
to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and
dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT 11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH 7.33 measured in the solvent control and at concentrations of 4500 µg/mL and 2250 µg/mL of test item
- Effects of osmolality: mOsm of 348 measured in the solvent control versus 295 at 4500 µg/mL and 325 at 2250 µg/mL
- Evaporation from medium: not examined
- Water solubility: Not indicated
- Precipitation:
Pre-experiment :
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. Phase separation was noted at 562.5 µg/mL and above with and without metabolic activation following 4 hours treatment. Following 24 hours treatment, phase separation occurred at 140.6 µg/mL and above.
Main experiments:
The culture medium was checked for precipitation or phase separation at the end of treatment. Phase separation was noted in the first experiment at 280 µg/mL and above with and at 280 µg/mL without metabolic activation. In the second experiment phase separation was observed at 560 µg/mL and above with metabolic activation.

- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration should be 10 mM, but not higher than 5 mg/mL or 5 µL/mL, unless limited by the solubility or toxicity of the test item.
RSG (Relative Suspension Growth) or RTG (Relative Total Growth) values (main experiment) below 50% are considered toxic. In case of toxic effects, the highest test item concentration of the main experiment should reduce the RSG or RTG value to approximately 10 - 20%.
The pre-experiment was performed in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation. Test item concentrations between 35.2 µg/mL and 4500 µg/mL (equal to a molar concentration of approximately 10 mM) were used. The highest concentration in the pre-experiment was chosen with regard to the purity (95.59%, preliminary information at the start of the experiment) and the molecular weight (430.618 g/mol) of the test item.
Toxic effects leading to RSG values below 50% were observed at 35.2 µg/mL and above in the absence and at 562.5 µg/mL and above in the presence of metabolic activation (4 hours treatment). After 24 hours treatment relevant toxic effects as described above occurred at 35.2 µg/mL and above.
The test medium was checked for precipitation or phase separation at the end of the treatment period (4 hours) before the test item was removed. Phase separation was noted at 562.5 µg/mL and above with and without metabolic activation following 4 hours treatment. Following 24 hours treatment, phase separation occurred at 140.6 µg/mL and above.
Both, pH value and osmolarity were determined in the pre-experiment at the two highest concentrations of the test item and in the solvent control without metabolic activation. There was no relevant shift of both parameters.
The dose range of the first and second experiment was set according to the cytotoxicity and solubility data generated in the pre-experiment. In both main experiments the individual concentrations were generally spaced by a factor of 2.0. A narrower spacing was used at the highest concentrations to cover the cytotoxic or phase separating range more closely.
To overcome problems with possible deviations in toxicity and solubility the main experiments were started with more than four concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant toxic effects indicated by a relative total growth of less than 50% were observed at 70 µg/mL and above in the first experiment without metabolic activation in culture II or at 140 µg/mL and above in culture II. In the second experiment without metabolic activation cytotoxic effects as described were noted at 17.5 µg/mL and above in culture I and at 35.0 µg/mL and above in culture II. In the second experiment with metabolic activation cytotoxicity occurred at 560 µg/mL and above with metabolic activation (culture I). The recommended cytotoxic range of approximately 10-20% relative total growth was covered in the second experiment without metabolic activation. In the presence of metabolic activation or after 4 hours treatment in the absence of metabolic activation, the dose range was limited by the solubility of the test item (phase separation).

Remarks on result:

other: strain/cell type: in vitro gene mutation assay with L5178Y cells

Remarks:

Migrated from field 'Test system'.

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with acetone		-	100.0	89	215	100.0	115	241
Pos. control with MMS	19.5	-	32.1	525	215	24.9	568	241
Test item	8.8	-	culture was not continued^#			culture was not continued^#
Test item	17.5	-	culture was not continued^#			culture was not continued^#
Test item	35.0	-	71.3	98	215	64.8	113	241
Test item	70.0	-	57.7	66	215	35.1	149	241
Test item	140.0	-	34.0	92	215	38.5	163	241
Test item	210.0	-	45.3	85	215	35.3	100	241
Test item	280 (PS)	-	34.6	139	215	26.2	135	241
Solv. control with acetone		+	100.0	88	214	100.0	104	230
Pos. control with CPA	3.0	+	77.2	225	214	55.5	268	230
Pos. control with CPA	4.5	+	49.8	286	214	41.8	504	230
Test item	70.0	+	culture was not continued^#			culture was not continued^#
Test item	140.0	+	106.5	112	214	89.3	169	230
Test item	280 (PS)	+	115.8	73	214	136.8	122	230
Test item	560 (PS)	+	102.1	140	214	86.2	141	230
Test item	840 (PS)	+	68.9	118	214	73.1	113	230
Test item	1120 (PS)	+	74.6	84	214	61.6	112	230
Experiment II / 24 h treatment			culture I			culture II
Solv. control with acetone		-	100.0	92	218	100.0	107	233
Pos. control with MMS	13.0	-	28.5	442	218	38.7	361	233
Test item	2.2	-	culture was not continued^#			culture was not continued^#
Test item	4.4	-	66.8	119	218	95.9	101	233
Test item	8.8	-	53.1	130	218	122.6	113	233
Test item	17.5	-	24.6	222	218	103.4	112	233
Test item	35.0	-	22.1	187	218	29.0	92	233
Test item	52.5	-	5.0	136	218	5.0	78	233
Test item	70.0	-	culture was not continued^##			culture was not continued^##
Experiment II / 4 h treatment			culture I			culture II
Solv. control with acetone		+	100.0	85	211	100.0	85	211
Pos. control with CPA	3.0	+	43.9	429	211	67.0	257	211
Pos. control with CPA	4.5	+	23.5	538	211	66.4	437	211
Test item	35.0	+	culture was not continued^#			culture was not continued^#
Test item	70.0	+	67.4	173	211	142.1	93	211
Test item	140.0	+	52.4	164	211	89.5	92	211
Test item	280.0	+	54.5	151	211	87.3	91	211
Test item	560 (PS)	+	46.1	126	211	89.2	102	211
Test item	840 (PS)	+	45.1	160	211	59.5	102	211

threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#   culture was not continued since a minimum of only four analysable concentrations is required

##   culture was not continued due to exceedingly severe cytotoxic effects

PS  phase separation

 

Conclusions:

Interpretation of results (migrated information):
negative

Under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of 1,1,4,4- tetramethylbutane- 1,4- diyl bis(2- ethylperoxyhexanoate) (CAS 13052- 09- 0) to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed in the absence of metabolic activation with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations with and without metabolic activation:

Experiment I

without S9 mix:                             35.0; 70.0; 140.0; 210.0; and 280.0 µg/mL
with S9 mix:                             140.0; 280.0; 560.0; 840.0; and 1120.0 µg/mL

Experiment II

without S9 mix:                                      4.4; 8.8; 17.5; 35.0; and 52.5 µg/mL
with S9 mix:                                      70.0; 140.0; 280.0; 560.0; 840.0 µg/mL

Relevant toxic effects indicated by a relative total growth of less than 50% were observed at 70 µg/mL and above in the first experiment without metabolic activation in culture II or at 140 µg/mL and above in culture II. In the second experiment without metabolic activation cytotoxic effects as described were noted at 17.5 µg/mL and above in culture I and at 35.0 µg/mL and above in culture II. In the second experiment with metabolic activation cytotoxicity occurred at 560 µg/mL and above with metabolic activation (culture I). The recommended cytotoxic range of approximately 10- 20% relative total growth was covered in the second experiment without metabolic activation. In the presence of metabolic activation or after 4 hours treatment in the absence of metabolic activation, the dose range was limited by the solubility of the test item (phase separation).

The culture medium was checked for precipitation or phase separation at the end of treatment. Phase separation was noted in the first experiment at 280 µg/mL and above with and at 280 µg/mL without metabolic activation. In the second experiment phase separation was observed at 560 µg/mL and above with metabolic activation.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both experiments.

In the first culture of the second experiment without metabolic activation an isolated increase of the mutation frequency exceeding the threshold of 126 above the corresponding solvent control occurred at an intermediate concentration of 17.5 µg/mL. This isolated increase was judged as irrelevant since no comparable increase was noted in the parallel culture under identical conditions or at higher concentrations in both parallel cultures. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11statistics software. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in the second culture of the second experiment without metabolic activation.This trend however, was judged as irrelevant since it actually was reciprocal, going down versus increasing concentrations. Another significantincrease was calculated in culture II of the second experiment in the presence of metabolic activation. This trend however, is considered a biologically irrelevant fluctuation since the mutation frequency did not exceed the threshold of 126 above the corresponding solvent control and it is not reproduced in the parallel culture.

In this study the range of the solvent controls was from 85 up to 115 mutant colonies per 10⁶cells; the range of the groups treated with the test item was from 66 up to 222 mutant colonies per 10⁶cells. The viability of the solvent control of the second culture of the first experiment with metabolic activation (63%) fell just short of the lower limit of 65%. This deviation was judged as irrelevant since it was very minor and the viability of the parallel culture remained well within the recommended range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 and 4.5 µg/mL) were used as positive controls and showed a distinct increase in induced total mutant colonies and an increase of the relative quantity of small versus large induced colonies with at least one of the concentrations.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Justification for selection of genetic toxicity endpoint

Ames + MLA + in vivo micronucleus, K1: The study was performed according to OECD guidelines and GLP.

Short description of key information:

The substance is positive for mutagenicity with metabolic activation in vitro in an Ames test and negative in a Mouse Lymphoma Assay.

In accordance with Endpoint Specific Guidance Chapter R.7A, Figure R.7.7-1 "Flow chart of the mutagenicity testing strategy", no further testing (i.e. no in vivo testing) need be proposed in the event of a negative mouse lymphoma assay or hprt assay, regardless to whether or not the gene mutation test in bacteria is positive or negative.  This therefore implies that when considering whether an in vivo gene mutation request is required for substances requiring Annex IX test proposals due to their volume bands, a negative mouse lymphoma assay or hprt assay is sufficient evidence to waive the need for an in vivo gene mutation test.

There is no in vitro cytogenicity study available however an in vivo micronucleus study is negative.

Endpoint Conclusion:No adverse effect observed (negative)

Link to relevant study records

Reference

Reference 1

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:

14 January 2003 - 23 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD guidelines and GLP.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, l'Arbresle, France
- Age at study initiation: on the day of treatment, the animals were approximately 6 weeks old.
- Weight at study initiation: no data
- Assigned to test groups randomly: yes, under following basis: by sex
- Fasting period before study: no
- Housing: by groups in polycarbonate cages
- Diet (e.g. ad libitum): ad libitum, A04 C pelleted maintenance diet (SAFE, Villemoisson-sur-Orge, France).
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the day of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30-70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 14 January 2003 - 23 February 2003

Route of administration:

intraperitoneal

Vehicle:

The vehicle was corn oil, batch Nos. 81K2204 and 062k0006 (Sigma, Saint-Quentin-Fallavier, France).

Details on exposure:

Route for the vehicle and the test substance: at the request of the Sponsor, the intraperitoneal route was used,
• Volume: 10 mL/kg,
• CPA: oral route, one treatment

Frequency of treatment:

Two treatments separated by 24 hours

Post exposure period:

24 hours

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

5 except for the high dose group where 8 animals were used

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Justification for choice of positive control(s): guideline recommendation
- Route of administration: Oral
- Doses / concentrations: Batch No. OD203A (Laboratoire Asta Médica, Mérignac, France) dissolved in distilled water at a concentration of 5 mg/mL. The preparation was stored at -20°C and thawed immediately before use.

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: In order to select the top dose-level for the cytogenetic study, 2000 mg/kg/day were administered twice, to three males and three females. The interval between each administration was 24 hours. Except for piloerection noted in both males and females, no clinical signs were noted. The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day. The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): 24 after the last treatment bone marrow samples were taken

DETAILS OF SLIDE PREPARATION: At the time of sacrifice, all the animals were killed by CO2 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

METHOD OF ANALYSIS: For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE). The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was Natalie Danford.

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance was also taken into account in the evaluation of data obtained.

Statistics:

When there was no significant within-group heterogeneity, using the heterogeneity chi-square test value (Lovell et al., 1989) (d), the frequencies of MPE in each treated group was compared with those in the concurrent vehicle control groups by using a 2 x 2 contingency table to
determine the χ2 value (Lovell et al., 1989) (d). When there was significant within-group heterogeneity, then that group was compared with the
control group using a non-parametric analysis, the Mann-Whitney test (Schwartz, 1969) (e). The student "t" test was used for the PE/NE ratio comparison. Probability values of p ≤ 0.05 was considered as significant.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

No clinical signs and no mortality were observed in the animals of both sexes given 500 and 1000 mg/kg/day. Piloerection was noted in both males and females at 2000 mg/kg/day.
For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group.
The PE/NE ratio was significantly (p < 0.001) lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.
Cyclophosphamide induced a highly significant increase (p < 0.001) in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Conclusions:

Interpretation of results (migrated information): negative
Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.

Executive summary:

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study.

In the main study, three groups of five male and five female mice received two intraperitoneal treatments at dose-levels of 500, 1000 or 2000 mg/kg/day, at a 24-hour interval.

One group of five males and five females received the vehicle (corn oil) under the same experimental conditions, and acted as control group.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg.

The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared.

For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The top dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no severe toxic effects were observed, the top dose-level was 2000 mg/kg/day.

The two other selected dose-levels for the main test were 500 and 1000 mg/kg/day. For both males and females, the mean values of MPE in the groups treated with the test item, were equivalent to those of the vehicle control group. The PE/NE ratio was significantly lower when compared to that of the vehicle control group, in the males treated at 2000 mg/kg/day, showing that the bone marrow cells were effectively exposed to the test item.

Cyclophosphamide induced a highly significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Under our experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of mice bone marrow cells

after two intraperitoneal administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.

Reference 2

Endpoint:

in vivo mammalian germ cell study: gene mutation

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Available data shows a negative outcome for genotoxicity based on these results, the data is conclusive but not sufficient for classification.