Formaldehyde, telomer with 1,3-benzenedimethanamine, 1,3-benzenediol and ethenylbenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Negative in 2 in vitro genotoxicity assays: bacterial reverse mutation assay and  mammalian in vitro mutagenicity assay (mouse lymphoma cells). A negative in vivo mouse micronucleus assay is available as well.

Link to relevant study records

Reference

Reference 1

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:

13-Mar-2012 to 15-May-2012

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

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

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:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 3, 10, 33, 100 and 333 µg/mL
Without S9-mix, 24 hours treatment: 3, 10, 33, 100 and 333 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 1, 10, 30, 50, 80, 90, 100 and 120 µg/mL
With S9-mix, 3 hours treatment: 1, 10, 30, 100, 150, 200, 250 and 350 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 3, 10, 30, 50, 70, 80, 90 and 100 µg/mL
With S9-mix, 3 hours treatment: 1, 3, 10, 100, 200, 300, 350 and 400 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide 10 µg/mL

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): 11 to 12 days

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

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (10^6) could be analysed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180 (24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 10^6 survivors, and for CP not below 700 per 10^6 survivors.

DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Statistics:

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 70 µg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and above in the absence of S9-mix at the 3 hour and prolonged treatment period. Precipitation in the exposure medium was observed at the dose level of 333 µg/mL in the presence of S9-mix..

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first experiment, HK 128 was tested up to cytotoxic levels of 85 and 97% in the absence and presence of S9-mix, respectively.

In the second experiment, HK 128 was tested up to cytotoxic levels of 87 and 73% in the absence and presence of S9-mix, respectively.

Conclusions:

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles. HK 128 is not mutagenic in the mouse lymphoma L5178Y test system

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 7.5- and 15 -fold for in the absence of S9-mix, and by 15- and 14-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, HK 128 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

 

In the presence of S9-mix, HK 128 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

standard strain from Prof. Bruce N. Ames, Berkeley, CA.

Species / strain / cell type:

S. typhimurium TA 97

Details on mammalian cell type (if applicable):

strain TA 97a from Prof. Bruce N. Ames, Berkeley, CA. his- frameshift Mutation at D6610, with rfa, uvrB and plcM101

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

standard strain from Prof. Bruce N. Ames, Berkeley, CA.

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

standard strain from Prof. Bruce N. Ames, Berkeley, CA.

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

standard strain from Prof. Bruce N. Ames, Berkeley, CA.

Metabolic activation:

with and without

Metabolic activation system:

rats treated with 500 mg/kg bw Aroclor 1254

Test concentrations with justification for top dose:

Without activation: 90, 30, 10, 3.3, 1.1 and 0.4 µg/plate.
With activation: 270, 90, 30, 10, 3.3 and 1.1 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-NOPD

Remarks:

for TA97a without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

for TA 97a with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

for TA98 and TA1535 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

Remarks:

for TA98, TA100 and TA1535 with activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive control substance:

sodium azide

Remarks:

for TA100 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: tBHPO

Remarks:

for TA102 without activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: DHA

Remarks:

for TA102 with activation

Details on test system and experimental conditions:

The exposure was performed according to the 'Plate Incorporation Assay', in which bacteria, test substance (and microsomes) are in contact on the plate without preceding incubation in the liquid state. The number of viable cells in the overnight-cultures is in the range of 2 x 108 cells per ml. For each sample the following solutions were combined: 0.1 ml of the overnight culture of the bacteria, 0.5 ml of S9-mix (or phosphate buffered saline for samples without metabolic activation), 0.1 ml of the appropriate test- or reference substance solution and 2 ml of top agar. The combined solutions were mixed and spread over a plate with minimal agar (9 cm diameter). After the top agar had solidified, the plates were incubated at 37 °C until the colonies were visible (2 days). The plates were counted manually or photographed with a video camera and the picture files were scanned for colonies by a computer program. The concentrations for the first experiment were set according to a preliminary toxicity test.

Evaluation criteria:

Means and standard deviation were calculated for the number of mutants in every concentration group. The criteria for a positive result are: A reproducible increase of the number of revertants to more than the following threshold values for at least one of the concentrations: For the strains with a low spontaneous revertant rate i.e. TA98 and TA1535: The 21/2 fold of the amount of the spontaneous revertants. For the strains with a high spontaneous revertant rate i.e. TA97a, TA100 and TA102: The 12/3 fold of the amount of the spontaneous revertants. These threshold values were derived from the variations in the control samples of our historic data of the Ames test.

Statistics:

Means and standard deviation were calculated for the number of mutants in every concentration group.

Key result

Species / strain:

S. typhimurium TA 97

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The concentrations for the first experiment were set according to a preliminary toxicity test. Toxicity was seen in the presence of S9-mix at concentrations of 200 µg/plate, in the absence of S9-mix at concentrations of 67 µg/plate and above. Therefore 270 µg/plate was chosen for the highest concentration with S9-mix and 90 µg/plate for the highest concentration without S9-mix. Both concentrations were assumed to be partly toxic to the bacteria, so 5 more lower concentrations, each one third of the preceding one, were used. The guidelines require 5 concentrations. In the main test, no toxicity was seen up to 90 µg/plate. At 270 µg/plate either microcolonies (single surviving bacteria from the background lawn which find enough histidine to form small colonies) were seen or there were some parts of the agar where no bacterial background lawn was seen. In the samples without a metabolising system, microcolonies were seen in all plates with 90 µg/plate. The lower concentrations were not toxic.

Conclusions:

The substance is not mutagenic in the Ames test, with or without metabolic activation, with the strains of Salmonella typhimurium TA97a, TA98, TA100, TA102 and TA1535 up to the limit of toxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo mouse micronucleus assay has not shown any potential for genotoxicity.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

NMRI BR mice (SPF) were used as test system. The animals were provided by Charles River, Sulzfeld, Germany. In the main test 5 male mice were treated per sampling time in each treatment group. Young adult animals were selected (6-7 weeks old). The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mice were identified by a unique number on the tail written with a marker pen. On arrival and at the start of the treatment, all animals were clinically examined to ensure selected animals were in a good state of health. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
The animals were housed in an air-conditioned room with approximately 15 air changes per hour and a controlled environment with a temperature of 21 ± 3°C (actual range 18.6 — 21.5°C) and a relative humidity of 30 — 70% (actual range 34 — 84%). Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the maximum level for humidity (with max. 14%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity. The room was illuminated with 12 hours artificial fluorescent light and 12 hours dark per day.
Group housing of 5 animals per sex per cage in labelled polycarbonate cages (type MII height:14 cm) containing Woody Clean bedding (Woody-Clean type 3/4; Technilab-BMI BV, Someren, The Netherlands). Paper bedding was provided as nest material (Technilab-BMI BV). Certificates of analysis were examined and then retained in the NOTOX archives.

Free access to standard pelleted laboratory animal diet (Altromin (code VRF 1), Lage, Germany) Certificates of analysis were examined and then retained in the NOTOX archives.
Free access to tap-water. Certificates of analysis (performed quarterly) were examined and then retained in the NOTOX archives.

Route of administration:

intraperitoneal

Vehicle:

Beckopox VEH 2626-Polymer was suspended in corn oil (Roth, Karlsruhe, Germany), Beckopox VEH 2626-Polymer concentrations were blended and treated with ultra-sonic waves to obtain a homogeneous suspension. Beckopox VEH 2626-Polymer concentrations were dosed within
2.5 hours after preparation.

Details on exposure:

The mice received an intraperitoneal injection of a maximum tolerated (high), an intermediate and a low dose of Beckopox VEH 2626-Polymer. 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. The systemic toxic signs were recorded at least once a day. The animals were weighed just prior to dosing. Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. Three dose groups, two comprising 1 male and 1 female, and one comprising 3 males and 3 females received a single dose of Beckopox VEH 2626-Polymer. The group comprising 3 males and 3 females were dosed with the highest concentration that was used for the main study. The study duration per dosing was three days. During this period mortality and physical condition were recorded at least once a day. In a dose range finding study ten animals (group A and B: 1 male and 1 female,
group C: 3 males and 3 females) were dosed intraperitoneally with 2000, 1500, and 750 mg/kg body weight (groups A, B and C, respectively).

In the main micronucleus study, five male mice were used per sampling time in each treatment group. The animals were dosed once and sampled at either 24 or 48 hours after test material administration.

Duration of treatment / exposure:

24 or 48 h

Frequency of treatment:

once

Post exposure period:

24 or 48 h

Remarks:

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

Remarks:

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

Remarks:

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

No. of animals per sex per dose:

5

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP;
CAS no. 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (Ziekenhuis Apotheek Noordoost-Brabant, Den Bosch, The Netherlands) dosed as a single intraperitoneal injection of 50 mg/kg body weight

Tissues and cell types examined:

Erythrocytes from bone marrow

Details of tissue and slide preparation:

Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing, The animals were sacrificed by cervical dislocation. 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 fetal calf serum (Invitrogen). 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 (Merck, Darmstadt, Germany) 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 (Merck) 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 (Klinipath, Duiven, The Netherlands) before they were embedded in MicroMount (Klinipath) and mounted with a coverslip.
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100x 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 1000x, 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. Observations/measurements in the study were recorded electronically using the following programme: REES Monitoring system version 1.5 (REES Scientific, Trenton, NJ, USA).

Evaluation criteria:

A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 1.3%0 ± 4.3%0 indicated are means for n=251).
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).
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.
The increase or decrease in the ratio polychromatic to normochromatic erythrocytes of the treatment groups compared to the ratio polychromatic to normochromatic erythrocytes of the negative control group is statistically evaluated using Wilcoxon Rank Sum Test (two sided test at p < 0.05).

Statistics:

Averages and standard deviations were calculated for polychromatic and normochromatic erythrocytes.
The increase or decrease in the ratio polychromatic to normochromatic erythrocytes of the treatment groups compared to the ratio polychromatic to normochromatic erythrocytes of the negative control group is statistically evaluated using Wilcoxon Rank Sum Test (two sided test at p < 0.05).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Toxicity was demonstrated in the high dose and the low dose. No changes in the PCE/NCE ratio were noted for the intermediate dose group.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Mice in all dose ranges displayed clinical signs of lethargy, rough coat and hunched posture.

Table 3 .      Mean number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes
	MALES
Group Treatment	Dose (mg/kg body weight)	Sampling time (hours)	Number of micronucleatedpolychromatic erythrocytes per 2000 polychromatic erythrocytes (Mean ± SD)	Ratio polychromatic/ normochromatic erythrocytes (Mean ± SD)
Solvent control= corn oil	0	24	0.2 ± 0.4	1.07 ± 0.07
Beckopox VEH 2626-Polymer	750	24	1.0 ± 1.4	0.69 ± 0.11*
Beckopox VEH 2626-Polymer	750	48	2.2 ± 1.9	0.52 ± 0.21*
Beckopox VEH 2626-Polymer	375	24	1.2 ± 1.1	0.93 ± 0.24
Beckopox VEH 2626-Polymer	190	24	0.0 ± 0.0	0.85 ± 0.14*
Positive control = CP	50	48	41.2 ± 15.2*	0.35 ± 0.22*

CP = cyclophosphamide

* Significantly different from corresponding control group (Wilcoxon Rank Sum Test, p < 0.05)

Conclusions:

The substance, when administered by intraperitoneal injection to mice at doses from 190 to 750 mg/kg bw, did not cause an increase in micronucleated polychromatic erythrocytes in bone marrow. The substance is negative for clastogenic effects in vivo, under conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

There was no evidence of genotoxic effects in the bacterial reverse mutation assay, nor in the mammalian in vitro mutagenicity assay in mouse lymphoma cells, nor in an in vivo mouse micronucleus assay. All assay results were negative (non-genotoxic).

Justification for selection of genetic toxicity endpoint
No evidence of genotoxic effects were observed in the bacterial reverse mutation assay, nor in the mammalian in vitro mutagenicity assay in mouse lymphoma cells, nor in an in vivo mouse micronucleus assay.

Justification for classification or non-classification

There was no evidence of genotoxic effects in the bacterial reverse mutation assay, nor in the mammalian in vitro mutagenicity assay in mouse lymphoma cells, nor in an in vivo mouse micronucleus assay. No classification is indicated.