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Genetic toxicity in vitro

Description of key information

In vitro gene mutation studies in bacteria and mammalian cells, and in vitro chromosome aberration studies were performed using BPA 2PO and BPA 5PO. Studies were performed according to OECD Guidelines or following methods similar to OECD Guidelines. All studies gave negative results in the presence and absence of metabolic activation.

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
october 1992-february 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: 2-aminoanthracene
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.
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Dianol 320 shows no mutagenic activity under the test conditions.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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 carrier 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 aberrations were recorded. Scoring followed the recommendations of the Ad Hoc Committee 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:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
from 200 µg/mL onwards +S9 and at 50 µg/mL -S9
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix (short- term treatment): 0, 12.5, 25, 50, 75, 100μg/mL
+S9 mix (short term treatment): 0, 25, 50, 100, 150, 200μg/mL
-S9 mix (continuous treatment, 24 and 48 hrs): 0, 6.25, 12.5, 25, 50, 100μg/mL
: The maximum concentration was established based on the growth inhibition test. In this test, more than 50% growth inhibition was observed at 93.75-750μg/mL for short-term treatment without S9, 187.5-750μg/mL with S9, and >93.75μg/mL for 24 hrs and 46.88μg/mL for 48 hrs continuous treatment. More than 1500μg/mL , the growth seemed to recover by deposition of test material,
Vehicle / solvent:
DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Short-term treatment without S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Short-term treatment with S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Continuous treatment
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: In short-term treatment cytotoxicity was observed at 75 and 100μg/mL without S9, and 150 and 200μg/mL with S9. In continuous treatment, at 100μg/mL.
Conclusions:
No increase in chromosomal aberration was observed either with short-term treatment (without S9 and with S9) or continuous treatment.
Executive summary:

No increase in chromosomal aberration was observed either with short-term treatment (without S9 and with S9) or continuous treatment.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
5 to 5000 ug per plate
Vehicle / solvent:
Demethyl sulphoxide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: aminoanthracene
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
negative with metabolic activation
negative without metabolic activation
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9: Rat liver, induced with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
-S9 mix: 0, 156, 313, 625, 1250, 2500, 5000μg/plate (TA100, TA1535, TA98, TA1537 and WP2 uvrA)
+S9 mix: 0, 156, 313, 625, 1250, 2500, 5000μg/plate (TA100, TA1535, TA98, TA1537 and WP2 uvrA)
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
Remarks:
Without S9 mix
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-AA
Remarks:
With S9 mix
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:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:


No mutagenic activity was found in the strain of S. typhimurium and E. coli and toxicity was not observed in any strain, with and without metabolic activation. Bisphenol A propoxylate is not mutagenic.
Executive summary:

To investigate the genetic toxicity of Bisphenol A propoxylated , gene mutation tests in bacteria were conducted with S. typhimurium TA 100, TA 1535, TA 98, TA 1537 and E. coli WP2uvrA strains.

No mutagenic activity was found in all strains of S. typhimurium and E. coli, with and without metabolic activation. Toxicity was not observed in any strain, with and without metabolic activation, up to the limit concentration.

Bisphenol A propoxylate is not mutagenic under the conditions of the test.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
November 1992 - April 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
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.
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
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, Dianol 320 did not show mutagenic activity at the HGPRT gene locus, both in the absence and in the presence of metabolic activating system.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate fraction (S9)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Solvent used is DMSO. Migrated to IUCLID6: In the absence of S9 mix.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Solvent used is DMSO. Migrated to IUCLID6: in the presence of S9 mix.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test substance did not have mutagenic potential under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

The in vitro genotoxicity of the registered substance was evaluated in accordance with REACH. It is concluded that it is not mutagenic and no in vivo testing is required.