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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A Bacterial reverse mutation assay with 4 Salmonella strains, an in vitro chromosome aberration study and in vitro mammalian cell gene mutation test are available with 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. All showed negative results, although in the chromosome aberration study an increase in polyploid cells was observed.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 1992 - April 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
GLP study from 1993, some deviations from current updated guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1984
Deviations:
yes
Remarks:
deviations according to updated guideline, such as number of cells; no purity indicated
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Target gene:
HGPRT locus
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
first assay: -S9: 0, 10, 25, 50, 75, 100, 150 µg/mL; +S9: 0, 10, 20, 40, 80, 100 µg/mL
second assay: -S9: 0, 25, 50, 75, 100, 125 µg/mL; +S9: 0, 25, 50, 75, 100, 125, 150 µg/mL
supplementary assay: -S9: 0, 130, 140, 150 µg/mL; +S9: 0, 160, 180, 200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Migrated to IUCLID6: 7,12-dimethylbenzanthracene (DMBA)
Details on test system and experimental conditions:
DURATION
- Preincubation period: 24h
- Exposure duration: 3h
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 cells/plate, 3 plates

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
Mutant frequency per 10E5 survivors = (no. of cells plated for PE/mean no. of PE colonies) x mean no. of 6-TG-r colonies. A comparison is made between the mutation frequency of the treated plaets and control plates. Statistical analysis is not applied.
The study is considered valid if:
solvent control data are acceptable and if positive control data are acceptable.
The test is considered positive if increases in mutation frequencies and/or mutatant colony numbers are consistently observed in each if the two assays. The significant elevantion of mutation frequency at only one concentration or in isolated single cultures at more than one concentration will be analysed on a case-by-case basis.
Statistics:
Statistical analysis is not applied.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 130 µg/mL onwards -S9, from 160 µg/mL onwards +S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: cloudiness was obserbed at 200 µg/mL


RANGE-FINDING/SCREENING STUDIES: included

In the first mutation assay no cytotoxicity was apparent at concentrations up to and including 100 µg/mL; total cell death was observed at 150 µg/mL (-S9). In the second test, no cytotoxicity was observed up to and including 150 µg/mL. In the supplementary assay, reduced plating efficiency was observed at and above 130 µg/mL (-S9); in the presense of S9 toxicity was observed at and above 160 µg/mL.

No increases in mutation frequencies (or mutant colony numbers) were observed in Dianol 320 treated culteres, both in the absence and presence of S9.

Conclusions:
Under the test conditions, 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) did not show mutagenic activity at the HGPRT gene locus, both in the absence and in the presence of metabolic activating system.
Executive summary:

4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was examined for mutagenic potential by measuring its ability to induce mutation in Chinese hamster (V79) cells at the hypoxanthine-guanine-phosphoribosyl transferase (HGPRT) locus. Under the experimental conditions of the test, 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) showed no evidence of mutagenic activity at the HGPRT gene locus, when cells were treated in the absence or presence of S-9 mix. The sensibility of the assay system was proven by the observed responses to known mutagens.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
october 1992-february1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study performed with 4 Salmonella test strains only; test substance indicated as Dianol 320 (but with appropriate CAS nr, certificate of analysis included.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli not tested
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
frameshift (TA 1537 and TA 98)
base substitution (TA 1535 and TA 100)
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (NADPH-generating system and hepatic microsomes from Aroclor 1254 treated rats).
Test concentrations with justification for top dose:
25-79-250-790-2500 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
2-nitrofluorene, sodium azide, 2-aminoanthracene, 9-aminoacridine,
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION

- Exposure duration: 2 days
Evaluation criteria:
Mean values of colony counts are calculated. A test material is considered to have mutagenic potential if it induces a dose-related increase in revertants over the concurrent solvent or vehicle controls, and if this increase reaches at least a doubling of the control values. If an increase of 2-fold or greater is recorded only at the highest tested dose (reproducible), this too is considered a positive result.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.
Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations in the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100. The assay was performed in two independent experiments both with and without liver microsomal activation. No substantial increase in revertant colony numbers of any of the tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). The test item is considered to be non-mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Study performed in 1993, not completely according to current updated guidelines.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
1983
Deviations:
yes
Remarks:
characterization of test and control substances not conducted according to the Standards; concentrations and homogeneity in carier are not confirmed; stability of the test and control substances are not determined. Only 100 metaphases per concentration
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
chromosome damage
Species / strain / cell type:
lymphocytes: human
Metabolic activation:
with and without
Metabolic activation system:
rat liver-derived metabolic activation system (S9 mix)
Test concentrations with justification for top dose:
without S9: 6.25, 12.5, 25.0, 50.0 µg/mL
with S9: 50.0, 100.0, 125.0, 150.0, 175.0, 200.0, 225.0 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ; DMSO
- Justification for choice of solvent/vehicle: no
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: chlorambucil
Details on test system and experimental conditions:
Slides prepared from cultures exposed to three concentrations only in the presence and absence of S9 mix at the 24h sampling time (and from cultures treated at the highest concentration selected for analysis in the presence of S9 mix at the 48h sampling time) were selected for chromosomal analysis.
The test was performed in duplicate.
Measured pH values of supernatants from control cultures and from selected Dianol 320 cultures showed no Dianol 320 treatment related effects on culture conditions.
Mitotic indices were scored from all cultures.
Slides from cultures treated with Dianol 320 at 50, 100, 150 and 175 µg/mL (24h sampling time) and at 150 and 175 µg/mL (48h sampling time) were selected for chromosomal analysis.
One hundred metaphases were scored from each culture (was not possible from one culture without S9 with Dioanol 320 at 175 µg/mL at 24h).
Cell division was arrested three hours before harvesting by the addition of colcemid to each culture.
Evaluation criteria:
On the basis of preliminary toxicity test data, the maximal test concentrations (- S9 and + S9) were selected as levels expected to result in approximately 50% depression in mitotic activity.
Assessment of toxicity: 1000 lymphocytes per culture.
At least 25 metaphases were scored from each negative and positive control cultures.
From 100 metaphases (with 46 centromeres), the chromosome number, all chromosomes normal or some aberrant, and specific types and numbers of aberrrations were recorded. Scoring followed the recommendations of the Ad Hoc Committe of the Environmental Mutagen Society and the Institute for Medical Research (1972).
Statistics:
The fische r Exact Probability test was used to compare two independent samples.
The one-tailed test was used to determine the frequency of aberrant metaphases for each treatment group compared with the corresponding solvent control group.
The difference between two groups is considered statistically significant if the p-value less than 0.05.
Species / strain:
other: human lymphocytes
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no disturbance of osmotic pressure

In the absence of S9 -mix, a significant increase in the highest dose tested (50 µg/mL) was observed only when gaps were included in the analysis; this effect was apparent only at a markedly toxic concentration; individual values were within or at the upper limit values of the historical control range.

In the presence of S9-mix, a statistical significant increase in aberrant cells was observed only in the presence of marked cytotoxicity (85% reduction in mitotic activity) at t=24h only (not at t=48h). The observed increases in aberrant cells under the described conditions are considered no evidence for clastogenic activity.

A marked increase in the incidence of polyploid cells was observed in Dianol 320 treated cells in the presence of S9 mix at and above 100 µg/mL (7-23-65 polyploid cells at 100 -150 -175 µg/mL, respectively) at a sampling time of 24h; at a sampling time of 48h, in the presence of S9 -mix, an increase in polyploid cells was observed at and above 150 µg/mL (8 -24 polyploid cells at 150 -175 µg/mL, respectively). This observation may be indicative of an effect of Dianol 320 on spindle formation/function.

Conclusions:
Based on the observed increases in aberrant cells only when gaps were included (-S9 mix) or in the presence of significant toxicity, Dianol 320 is considered to be not clastogenic. In the presence of S9-mix an increase in aberrant cells was only shown in the presence of marked cytotoxicity, thus it was concluded that the substance did not show evidence of clastogenic activity. However, since an increase in the incidence of polyploid cells was observed in the presence of S9-mix (both at 24h and at 48h), Dianol may have the potential to disturb mitotic processes and cell cycle progression.
Executive summary:

The effects on chromosaomal structure of exposure of 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) were investigated in cultured human lymphocytes. Tests were conducted with and without the inclusion of a rat liver-derived metabolic activation system (S-9 mix). Without S-9 mix cells were exposed continously for 24 hoursn with S-9 mix exposure was limited to three hours and cells were harvested 21 or 45 hours later.

Based on the observed increases in aberrant cells only when gaps were included (-S9 mix) or in the presence of significant toxicity, 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) is considered to be not clastogenic. In the presence of S9-mix an increase in aberrant cells was only shown in the presence of marked cytotoxicity, thus it was concluded that the substance did not show evidence of clastogenic activity. However, since an increase in the incidence of polyploid cells was observed in the presence of S9-mix (both at 24h and at 48h), the substance may have the potential to disturb mitotic processes and cell cycle progression.

In the absence of S9 -mix, a significant increase in the highest dose tested (50µg/mL) was observed only when gaps were included in the analysis; this effect was apparent only at a markedly toxic concentration; individual values were within or at the upper limit values of the historical control range.

In the presence of S9-mix, a statistical significant increase in aberrant cells was observed only in the presence of marked cytotoxicity (85% reduction in mitotic activity) at t=24h only (not at t=48h). The observed increases in aberrant cells under the described conditions are considered no evidence for clastogenic activity.

A marked increase in the incidence of polyploid cells was observed in 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) treated cells in the presence of S9 mix at and above 100µg/mL (7-23-65 polyploid cells at 100 -150 -175 µg/mL, respectively) at a sampling time of 24h; at a sampling time of 48h, in the presence of S9 -mix, an increase in polyploid cells was observed at and above 150 µg/mL (8 -24 polyploid cells at 150 -175 µg/mL, respectively). This observation may be indicative of an effect of 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) on spindle formation/function.

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

Genetic toxicity in vivo

Description of key information

1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was then tested in a Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow : no clastogenic or aneugenic effects were observed.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-Nov-2013 to 23-Jan-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
July 21, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
May 31, 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 34.3 ± 2.1 g and the range was 30 - 40 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 21.9°C
- Humidity (%): 38 - 58%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: propylene glycol
The solid test substance was crushed and ground in a mortar with pestle to improve the consistency.
4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was dissolved in propylene glycol. 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) concentrations were treated with ultra-sonic waves until 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) had completely dissolved.

- Justification for choice of solvent/vehicle: Test compound was soluble in propylene glycol. Propylene glycol has been accepted and approved by authorities and international guidelines

- Concentration of test material in vehicle: 100, 200 and 400 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 5 ml/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The solid test substance was crushed and ground in a mortar with pestle to improve the consistency. 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was dissolved in propylene glycol (Merck, Darmstadt, Germany). The specific gravity of propylene glycol is 1.036 g/ml. 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) concentrations were treated with ultra-sonic waves until 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) had completely dissolved. 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) concentrations were dosed within 3 hours after preparation.
Duration of treatment / exposure:
The animals treated with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) and vehicle control were dosed twice with a 24 hours interval and were sacrificed 24 hours after the last treatment. Positive control animals received a single dose and were sacrificed 24 hours after.
Frequency of treatment:
The animals treated with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) and vehicle control were dosed twice (with a 24 hours interval).
Positive control animals received a single dose.
Remarks:
500, 1000 and 2000 mg/kg body weight
No. of animals per sex per dose:
At least five animals per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control(s): Has been accepted and approved by authorities and international guidelines
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Vinblastin
- Justification for choice of positive control(s): Has been accepted and approved by authorities and international guidelines
- Route of administration: Oral
- Doses / concentrations: 9 mg/kg body weight
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

ISOLATION OF BONE MARROW
Bone marrow of all groups was sampled 48 hours after the first 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. Each bone was flushed with approximately 1 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands).

PREPARATION OF BONE MARROW SMEARS FOR MICRONUCLEI ANALYSIS
The cell suspension of one bone was collected and centrifuged at 216 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 remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the 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.

PREPARATION OF BONE MARROW SMEARS FOR FISH ANALYSIS
Bone marrow suspension of the other bone was processed through a cellulose column containing 0.8 g of a 50:50 mixture of cellulose type 50 (Sigma-Aldrich Chemie GmbH) and alpha-cellulose (Sigma-Aldrich Chemie GmbH) (one column per animal) and using 4 ml fetal bovine serum as the eluate. Eluted cells were centrifuged at 216 g for 5 min and 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 of the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v) ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. The slides were marked with the 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 (prepared at relatively high cell density) were stored at =-15ºC for potential FISH analysis.
STAINING OF THE BONE MARROW SMEARS FOR MICRONUCLEUS EVALUATION
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene (Klinipath, Duiven, The Netherlands)/pertex (Klinipath) and mounted with a coverslip in an automated coverslipper (Leica Microsystems B.V., Rijswijk, The Netherlands).

ANALYSIS OF THE BONE MARROW SMEARS FOR MICRONUCLEI
To prevent bias, all slides were randomly coded before examination. An adhesive label with the study identification number and code was stuck over the marked slide. 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 at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.


DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of 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.
Evaluation criteria:
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, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.
Statistics:
Wilcoxon Rank Sum Test, one-sided, p < 0.05
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
The animals showed the following toxic signs: rough coat (2 males, 1 female) and lethargy (1 male, 1 female). Within 22 hours after dosing these animals had recovered from the treatment. No treatment related signs of toxicity were observed in 1 male and 2 female animals.


RESULTS OF DEFINITIVE STUDY

- Clinical signs of toxicity in test animals:
No treatment related clinical signs or mortality were noted in any animal treated with 4, 4’-Isopropylidenediphenol propoxylated (BPA+2PO) or control animals receiving vehicle, cyclophosphamide or vinblastin with the exception of 2 animals dosed with 2000 mg 4, 4’-Isopropylidenediphenol propoxylated (BPA+2PO) /kg body weight which showed lethargy within 2.5 hours after the second dosing. Both animals had recovered from the treatment within 24 hours after the second dosing
- Induction of micronuclei (for Micronucleus assay):
No biologically relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with test substance.
- Ratio of PCE/NCE (for Micronucleus assay):
No decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. However, the clinical signs observed were taken to indicate that systemic absorption had occurred
- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.

Mean number of micronucleated polychromatic erythrocytes

and ration of polychromatic/normochromatic erythrocytes

Group

treatment

Dose (mg/ kg BW)

Sampling time (hours)

Number of micronucleated polychromatic erythrocytes

(mean±SD)(1,2)

Ratio polychromatic/

normochromatic erythrocytes

(mean±SD)(1,3)

A

Vehicle control

0

48

1.8±0.4

0.97± 0.01

B

Test substance

2000

48

2.4± 1.5

0.85± 0.06

C

Test substance

1000

48

2.6± 1.7

0.86± 0.19

D

Test substance

500

48

2.4± 0.5

0.90± 0.08

E

CP

40

48

20.6± 3.3(4)

0.68± 0.15

F

Vinblastin

9

48

32.2± 4.7(4)

0.30± 0.07

 

Test substance = 4,4’-Isopropylenediphenol propoxylated

Vehicle control = propylene glycol

CP= cyclophosphamide

(1)  Five animals per treatment group

(2)  At least 2000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.

(3)  The ratio was determined from at least the first 1000 erythrocytes counted.

(4)  Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P= 0.01).

 

 

 

 

 

INDIVIDUAL DATA

Individual data (males)

(group A : oral intubation of the vehicle)

(Group B : oral intubation of 4,4’-Isopropylenediphenol propoxylated at 2000 mg/kg body weight)

(Group C : oral intubation of 4,4’-Isopropylenediphenol propoxylated at 1000 mg/kg body weight)

(Group D : oral intubation of 4,4’-Isopropylenediphenol propoxylated at 500 mg/kg body weight)

(Group E : oral intubation of cyclophosphamide at 40 mg/kg body weight)

(Group F : oral intubation of vinblastin at 9 mg/kg body weight)

 

Group

Animal number

Number of micronucleated polychromatic erythrocytes

Number of polychromatic erythrocytes scored for micronuclei(1)

Number of polychromatic erythrocytes

Number of normochromatic erythrocytes(1)

Ratio polychromatic/

normochromatic erythrocytes(1)

A

1

1

2089

520

532

0.98

A

2

2

2019

504

510

0.99

A

3

2

2003

491

513

0.96

A

4

2

2027

512

527

0.97

A

5

2

2001

499

509

0.98

 

 

 

 

 

 

 

B

6

2

2045

478

522

0.92

B

7

5

2043

482

529

0.91

B

8

1

2015

472

568

0.83

B

9

2

2007

446

554

0.81

B

10

2

2005

458

591

0.77

 

 

 

 

 

 

 

C

11

1

2069

509

520

0.98

C

12

1

2025

503

514

0.98

C

13

3

2011

464

555

0.84

C

14

5

2013

352

648

0.54

C

15

3

2003

511

524

0.98

 

 

 

 

 

 

 

D

16

3

2003

467

544

0.86

D

17

3

2013

505

543

0.93

D

18

2

2005

513

503

1.02

D

19

2

2035

483

570

0.85

D

20

2

2009

463

557

0.83

 

 

 

 

 

 

 

E

21

19

2011

471

529

0.89

E

22

19

2082

427

579

0.74

E

23

17

2022

344

698

0.49

E

24

23

2039

389

642

0.61

E

25

25

2007

410

610

0.67

 

 

 

 

 

 

 

F

26

36

2011

206

821

0.25

F

27

26

2011

285

721

0.40

F

28

37

2033

219

848

0.26

F

29

29

2097

263

743

0.35

F

30

33

2093

201

814

0.25

 

(1) The ratio was determined from the first 1000 erythrocytes counted.

Conclusions:
It is concluded that this test is valid and that 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.
Executive summary:

4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) was tested in the Micronucleus Test in mice (OECD 474), to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow.

The test substance was dissolved in propylene glycol.

In the dose range finding test, in total three male and three female animals were dosed with 2000 mg 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) per kilogram body weight. The animals showed the following toxic signs: rough coat (2 males, 1 female) and lethargy (1 male, 1 female). Within 20 hours after dosing these animals had recovered from the treatment. No treatment related signs of toxicity were observed in 1 male and 2 female animals. Since there were no substantial differences between sexes in toxicity, only males were used in the main study.

In the main study male animals were dosed on two consecutive days via oral gavage with vehicle or with 2000, 1000 and 500 mg 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) per kg body weight. Two positive control groups were dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight and 9 mg vinblastin per kg body weight, respectively. In total 6 treatment groups were used, each consisting of 5 animals.

No treatment related clinical signs or mortality were noted in any animal treated with 4, 4’-Isopropylidenediphenol propoxylated (BPA+2PO) or control animals receiving vehicle, cyclophosphamide or vinblastin with the exception of 2 animals dosed with 2000 mg 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) /kg body weight which showed lethargy within 2.5 hours after the second dosing. Both animals had recovered from the treatment within 24 hours after the second dosing.

Bone marrow of all groups was sampled 48 hours after the first dosing.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide and vinblastin, the positive control substances, induced statistically significant increases in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide and vinblastin showed expected decreases in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that 4,4’-Isopropylidenediphenol, propoxylated (BPA+2PO) is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

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

Additional information

All three in vitro studies required were performed according to OECD test guidelines. In all tests toxicity was observed at concentrations tested with and without metabolic activation. Substance 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol shows no mutagenic activity in a bacterial reverse mutation assay with Salmonella strains, with and without metabolic activation. Under the test conditions, 1,1'-isopropylidenebis(p-phenyleneoxy)dipropan-2 –ol did not show mutagenic activity at the HGPRT gene locus, both in the absence and in the presence of metabolic activating system. Based on the observed increases in aberrant cells only when gaps were included (-S9 mix) or in the presence of significant toxicity, 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol is considered to be not clastogenic. In the presence of S9-mix an increase in aberrant cells was only shown in the presence of marked cytotoxicity, thus it was concluded that the substance did not show evidence of clastogenic activity. However, since an increase in the incidence of polyploid cells was observed in the presence of S9-mix (both at 24h and at 48h), 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol may have the potential to disturb mitotic processes and cell cycle progression.

Thus, 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol was then tested in a Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. The study procedures used were based on the most recent OECD and EC guidelines.

In the dose range finding test, in total three male and three female animals were dosed with 2000 mg/kg of 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol dissolved in propylene glycol. The animals showed the following toxic signs: rough coat (2 males, 1 female) and lethargy (1 male, 1 female). Within 20 hours after dosing these animals had recovered from the treatment. No treatment related signs of toxicity were observed in 1 male and 2 female animals. Since there were no substantial differences between sexes in toxicity, only males were used in the main study.

In the main study male animals were dosed on two consecutive days via oral gavage with vehicle or with 2000, 1000 and 500 mg/kg of 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol. Two positive control groups were dosed once via oral gavage with 40 mg/kg cyclophosphamide (CP)or 9 mg/kg vinblastin. In total 6 treatment groups were used, each consisting of 5 animals.

No treatment related clinical signs or mortality were noted in any animal treated with 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol or control animals receiving vehicle, cyclophosphamide or vinblastin with the exception of 2 animals dosed with 2000 mg/kg of 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol which showed lethargy within 2.5 hours after the second dosing. Both animals had recovered from the treatment within 24 hours after the second dosing.

Bone marrow of all groups was sampled 48 hours after the first dosing.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide and vinblastin, the positive control substances, induced statistically significant increases in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The groups that were treated with cyclophosphamide and vinblastin showed expected decreases in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that 1,1-isopropylidenebis(p-phenyleneoxy)dipropan-2-ol is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Justification for classification or non-classification

The available data do not lead to classification for genotoxicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.