Reaction mass of 4,4'-isopropylidenediphenol and phenol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the properties of the target substance Reaction mass of phenol and 4,4’-isopropylidenediphenol can be predicted by studies conducted with the source substances phenol, 4,4’-isopropylidenediphenol (BPA), and 2-acetone, polymer with phenol, because the target substance Reaction mass of phenol and 4,4’-isopropylidenediphenol contains phenol (40-45%, typical concentration ca. 40%) and 4,4’-isopropylidenediphenol (BPA) (20-40%, typical concentration ca. 33%) as main constituents. Both constituents are data rich substances with distinct hazard properties, so that mainly data on the constituents have been applied to characterize the Reaction mass of phenol and 4,4’-isopropylidenediphenol. Since this is a common approach in mixture hazard assessment, is reasonable to apply it also to multi-constituent substances.
Additionally, some data from a structurally related substance (2-acetone, polymer with phenol) containing the same constituents/impurities at different concentrations are available, which are applied to characterize the environmental fate and ecotoxicity of the impurities present in the Reaction mass of phenol and 4,4’-isopropylidenediphenol.

This read-across hypothesis corresponds to scenario 2 - different compounds have qualitatively and quantitatively the same type of effects - of the read-across assessment framework i.e. properties of the target substance Reaction mass of phenol and 4,4’-isopropylidenediphenol are predicted to be similar to those of the source substances phenol, 4,4’-isopropylidenediphenol (BPA), and 2-acetone, polymer with phenol.

Therefore, read-across from the available studies with the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance Reaction mass of phenol and 4,4’-isopropylidenediphenol, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
please refer to justification for read-across attached to Iuclid section 13

3. ANALOGUE APPROACH JUSTIFICATION
please refer to justification for read-across attached to Iuclid section 13

4. DATA MATRIX
please refer to justification for read-across attached to Iuclid section 13

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

read-across: supporting information

Cross-reference 2

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study which meets basic scientific principles. BPA is a constituent of the reaction mass so that its hazard data are relevant for the assessment of the reaction mass.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Experiment 1: Oocytes from untreated MF1 mice (n=4-12 per group) were matured in vitro with 0, 50, 100, 200, 400, or 800 ng/ml, 4 or 10 µg/ml BPA (0.22-43.8 µM) and evaluated for meiotic progression.
Experiment 2: C57Bl x CBA/Ca F1 hybrid female mouse pups at 22 days of age (n=4 per group, usually) were administered by gavage 0, 20, 40, or 100 ng/g BPA on 7 consecutive days. Oocytes were isolated and matured ex vivo, then evaluated for meiotic progression.

GLP compliance:

no

Type of assay:

other: assessment of meiotic progression

Species:

mouse

Strain:

other: MF1; C57Bl x CBA/Ca hybrid

Sex:

female

Route of administration:

other: in vitro; oral gavage

Vehicle:

corn oil

Sex:

female

Genotoxicity:

negative

Remarks on result:

other: Experiment 1: Bisphenol A did not increase hyperploidy at meiosis II at any tested concentration.

Sex:

female

Genotoxicity:

negative

Remarks on result:

other: Experiment 2: Bisphenol A exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Additional information on results:

Experiment 1: At the highest BPA dose (10 ug/ml), meiotic progression, nuclear maturation, and chromosomal constitution were increased. This dose also affected spindle formation, distribution of pericentriolar material, and chromosome alignment on the spindle, causing significant meiotic arrest. BPA did not increase hyperploidy at meiosis II at any tested concentration.
Experiment 2: BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Experiment 1: BPA did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Conclusions:

Experiment 1: BPA did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour.

Executive summary:

Experiment 1: Oocytes from untreated MF1 mice (n=4-12 per group) were matured in vitro with 0, 50, 100, 200, 400, or 800 ng/ml, 4 or 10 ug/ml BPA (0.22-43.8 µM) and evaluated for meiotic progression. At the highest BPA dose (10 µg/ml), meiotic progression, nuclear maturation, and chromosomal constitution were increased. This dose also affected spindle formation, distribution of pericentriolar material, and chromosome alignment on the spindle, causing significant meiotic arrest. BPA did not increase hyperploidy at meiosis II at any tested concentration.

Experiment 2: C57Bl x CBA/Ca F1 hybrid female mouse pups at 22 days of age (n=4 per group, usually) were administered by gavage 0, 20, 40, or 100 ng/g BPA on 7 consecutive days. Oocytes were isolated and matured ex vivo, then evaluated for meiotic progression. BPA exposure was not associated with any significant effects on meiotic progression, spindle abnormalities, or aberrant chromosome behaviour. The authors concluded that low chronic BPA exposure in vivo does not appear to pose a risk for induction of errors in chromosome segregation at first meiosis in mouse oocytes.

Cross-reference 3

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is comparable with OECD Guideline 474 with acceptable restrictions (partly limited documentation, e.g. no details about test substance; only one dose tested; no historical control data; no concurrent positive control [but in parallel experiments]; no data about clinical signs of toxicity; PCE/NCE ratio determined only in 100 cells). Phenol is a constituent of the reaction mass so that phenol hazard data are applied in the hazard assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

(one dose)

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

Age of 10-16 weeks; groups of mice of the same age selected, body weight within 2 g of a common weight.
No further data available.

Route of administration:

intraperitoneal

Vehicle:

phosphate-buffered saline (PBS)
solutions prepared within 2 h prior to administration

Details on exposure:

Mice i.p. injected with PBS or 300 mg/kg bw phenol in PBS (volume 0.4 ml in each group) and 26 h later animals killed by cervical dislocation, femurs removed and cell samples obtained.

Duration of treatment / exposure:

single i.p. injection

Frequency of treatment:

Once

Post exposure period:

26 h

Remarks:

Doses / Concentrations:
0 or 300 mg/kg bw
Basis:
other: actual injected

No. of animals per sex per dose:

6 males

Control animals:

yes, concurrent vehicle

Positive control(s):

0.2, 1.0, 2.0 mg/kg bw mitomycin C (n=4-6) in parallel experiments

Tissues and cell types examined:

polychromatic (PCE) and normochromatic erythrocytes (NCE) in bone marrow

Details of tissue and slide preparation:

Bone marrow aspirates centrifuged and cell pellet resuspended; smear prepared on slide and air-dried, fixed in absolute methanol and stored before staining with acridine orange; number of PCEs containing micronuclei & number of micronuclei in each PCE recorded in 2000 PCEs; coded slides; %PCEs determined among 100 erythrocytes.

Evaluation criteria:

No data

Statistics:

no data

Sex:

male

Genotoxicity:

other: weak positive effects at myelotoxic dose level

Toxicity:

yes

Remarks:

(decreased PCE/NCE ratio; no further data)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

% PCE in vehicle control 50.0+-4.8 (mean +- S.E.) and in treated mice 19.8+- 2 4.
Micronucleated PCE per 1000 PCE: 3.3+-0.2 in control and 11.4+- 1.9 in treated mice.
Similar results were obtained using the same bone marrow samples but following cellulose column separation of samples before staining the cells.

No data are available on clinical signs of toxicity but severe effects are expected at this dose level including hypothermia (compare with Spencer et al. 2007 in the same Section).

Conclusions:

Interpretation of results: weak positive at myelotoxic dose level
The i.p. injection of 300 mg/kg bw induced clear myelotoxic effects (decreased PCE/NCE ratio) and weak positive effects in the mouse bone marrow micronucleus assay.

Executive summary:

The study is comparable with OECD Guideline 474 with acceptable restrictions (partly limited documentation, e.g. no details about test substance; only one dose tested; no historical control data; no concurrent positive control [but in parallel experiments]; no data about clinical signs of toxicity; PCE/NCE ratio determined only in 100 cells).

In this mouse bone marrow micronucleus test 6 male B6C3F1 mice per group received a single i.p. injection of 0 or 300 mg/kg bw phenol. Bone marrow samples were prepared 26 h after application. Concerning the cytotoxic effects in bone marrow the % PCE (polychromatic erythrocytes) decreased from 50.0+-4.8 (mean +- S.E.) in vehicle control to 19.8 +- 2 4 in treated mice. A 3.3-fold increase was found in the micronucleated PCE per 1000 PCE: 3.3 +-0.2 in control and 11.4 +- 1.9 in treated mice.

Conclusion: The i.p. injection of 300 mg/kg bw induced a weak positive effects in the mouse bone marrow micronucleus assay combined with clear myelotoxic effects (decreased PCE/NCE ratio).

Cross-reference 4

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study is comparable to OECD 474 with acceptable restrictions (party limited documentation, e.g. no details on test substance, vehicle, concentration of phenol in vehicle; data taken from a poster presented at the 42nd Annual Meeting of the Society of Toxicology, 09. - 13.03.2003 in Salt Lake City, Utah, USA) Phenol is a constituent of the reaction mass so that phenol hazard data are applied in the hazard assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

8 week old male & female CD1 mice, housed in standard environment or a supported environment.

Standard environment
Housing: hanging wire cages
Room temperature/humidity: 21-23°C/40-60%

Thermoregulatory support conditions
Housing: plastic “shoebox” style cages with corn cob bedding & a small nesting hut, warming blanket at 30°C placed under half of the cage
Room temperature/humidity: 28-30°C/27-47%

No further data

Route of administration:

intraperitoneal

Vehicle:

no data

Details on exposure:

No details

Duration of treatment / exposure:

single injection

Frequency of treatment:

sigle application

Post exposure period:

24 or 48 h

Remarks:

Doses / Concentrations:
0 or 300 mg/kg bw
Basis:
no data

No. of animals per sex per dose:

Data on MN induction: not documented (but presumably surving mice, see Table below on mortality).
Data on mortality: 10 mice per sex in control and 24 mice per sex in treated group.

Control animals:

yes, concurrent vehicle

Positive control(s):

120 mg/kg bw cyclophosphamide monohydrate (only males)

Tissues and cell types examined:

Body temperature measured 0, 24, 48 h after application.
Mortality rate recorded.
MN per/1000 PCE determined in preparations of the bone marrow tissue 24 or 48 h after application.

Details of tissue and slide preparation:

No details

Evaluation criteria:

No data

Statistics:

Statistical analysis performed (no details except level of significance, a<0.05).

Sex:

male/female

Genotoxicity:

positive

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Body Temperature (BT) of CD1 Mice after Phenol Treatment
Standard Environment: in males and females the BT decreased to ca. 31.5°C 24 h after application of 300 mg/kg bw; BT of 29°C in males and ca. 30.5 in females were measured 48 h after application. At the same time only a slight decrease in BT of ca. 1°C was detected in male and female mice using thermoregulative support.

Mortality
A sgnificant increase in lethality in phenol-treated mice with thermoregulatory support was observed (see Table below) which might indicate a protective role of hypothermia against lethality.

Micronuclei (MN) induction
Thermoregulatory support did not prevent MN induction in phenol-treated mice at 24 h post-dosing but at 48 h post-dosing.
The frequency of MN-PCE increased at 48 h as compared to the frequency at 24 h in phenol-treated unsupported mice. A slight but not significant increase in MN occurred in the thermoregulatory supported control male mice at 24 h but not 48 h. BT measurement at 0, 24 and 48 h did not allow full characterization of BT profiles.

Mortality in phenol-treated mice

Dose in mg/kg bw	Mortality in males	Mortality in females
	Unsupported thermoregulation
0	0/10	0/10
300	5/24	1/24
-	Supported thermoregulation
0	1/10	0/10
300	15/24	14/24

MN formation in phenol-treated mice

Dose in mg/kg bw (sex)	Post exposure interval in hours	MN per/1000 PCE
		Unsupported	Supported
0 (m)	24	2.1 ± 1.8	5.0 ± 3.1
300 (m)	24	10.8 ± 8.5*	9.3 ± 4.4*
0 (f)	24	2.5 ± 2.0	3.6 ± 1.0
300 (f)	24	11.3 ± 9.3*	20.6 ± 7.4*
0 (m)	48	1.1 ± 0.4	3.2 ± 2.0
300 (m)	48	18.3 ± 1.8*	3.0 ± 2.4
0 (f)	48	2.4 ± 1.4	1.8 ±1.2
300 (f)	48	17.8 ± 14.3*	6.1 ± 3.1
Positive control	24	79.9 ± 20.1	-

*: alpha <= 0.05

Conclusions:

Interpretation of results: threshold dependent chromosome mutagenic activity
Thermoregulatory support prevented the induction of mirconuclei measured 48 h after application of phenol. Initial sustained hypothermia in phenol-treated animals was not prevented by thermoregulatory support and likely accounts for the increase in micronuclei at 24 h.
Authors comment: Distinguishing the role of hypothermia in the induction of micronuclei from direct action of phenol/metabolites with the cellular target(s) by providing thermoregulatory support is unlikely.

Executive summary:

Study is comparable to OECD 474 with acceptable restrictions (party limited documentation).

The authors evaluated the effects of thermoregulatory support on the induction of micronuclei in the bone marrow of phenol-treated mice. Doses of phenol that did not induce substantial hypothermia in mice were not associated with an increase frequency of micronuclei ( see Spencer et al., 2007, in this Section). It should be shown in this study that the prevention of hypothermia will inhibit the induction of micronuclei in the bone marrow of phenol-treated mice. Mice housed in standard environment or under thermoregulatory support conditions received a single injection of i.p. 0 or 300 mg/kg bw and bone marrow was prepared 24 or 48 h after application. In unsupported males and females the body temperature (BT) decreased to ca. 31.5°C 24 h after application; BT of 29°C in males and ca. 30.5 in females were measured 48 h after application. Only a slight decrease in BT of ca. 1°C was detected at the same time in male and female mice using thermoregulative support. The mortality rate was increased in supported mice. Thermoregulatory support did not prevent MN induction in phenol-treated mice at 24 h post-dosing but at 48 h post-dosing. BT measurement at 0, 24 and 48 h did not allow full characterization of BT profiles. Therefore, additional experiments were conducted on the effectiveness of thermoregulatory support (BT measured every 5 minutes). Also in mice receiving thermoregulatory support a dose of 300 mg/kg bw resulted in an initial decrease in BT (min. 32.5°C) reaching average BT again after approx. 3 h.

Conclusion: Thermoregulatory support prevented the induction of mirconuclei measured 48 h after application of phenol. Initial sustained hypothermia in phenol-treated animals was not prevented by thermoregulatory support and likely accounts for the increase in micronuclei at 24 h.

Cross-reference 5

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

: oocyte development

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study which meets basic scientific principles. BPA is a constituent of the reaction mass so that its hazard data are relevant for the assessment of the reaction mass.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Female C57BL/6J mice were fed one of two diets for at least one week prior to mating: 1) TestDiet AIN-93G, a casein diet that does not contain soy as a protein source but does contain soybean oil, and 2) Harlan Teklad Sterilizable Rodent Diet 8656, a soy-based diet. At 21 days of age, female offspring were treated orally with BPA (20, 40, 100, 200, or 500 µg/kg-day) for 7 days and effects on oocyte development were determined.

GLP compliance:

no

Type of assay:

other: assessment of meiotic progression

Species:

mouse

Strain:

C57BL

Sex:

female

Route of administration:

oral: gavage

Vehicle:

corn oil

Additional information on results:

Abnormalities of metaphase II were observed in 2% of the eggs from the offspring of control group females on the casein diet, as compared to 8% of control group offspring of animals on the soy diet. The casein diet produced an apparent linear dose response, with increases in spindle/chromosome alignment abnormalities at the 200 ug/kg-day BPA dose level. The soy diet produced an apparent U-shaped dose response. Offspring from the 500 ug/kg-day dose group on the soy diet had a higher abnormal metaphase II rate than that of the combined data for the vehicle and baseline (no vehicle, no BPA dosing) control groups, but no difference was seen for this group as compared with either control group individually.

The authors could not replicate their initial findings on “congression failure” (Hunt et al. 2003. Curr Biol. 13: 546 – 553) and report significant differences between the two diets investigated.

Conclusions:

The authors could not replicate their initial findings on “congression failure” (Hunt et al. 2003. Curr Biol. 13: 546 – 553) and report significant differences between the two diets investigated.

Executive summary:

Female C57BL/6J mice were fed one of two diets for at least one week prior to mating: 1) TestDiet AIN-93G, a casein diet that does not contain soy as a protein source but does contain soybean oil, and 2) Harlan Teklad Sterilizable Rodent Diet 8656, a soy-based diet. At 21 days of age, female offspring were treated orally with BPA (20, 40, 100, 200, or 500 µg/kg-day) for 7 days and effects on oocyte development were determined. Abnormalities of metaphase II were observed in 2% of the eggs from the offspring of control group females on the casein diet, as compared to 8% of control group offspring of animals on the soy diet. The casein diet produced an apparent linear dose response, with increases in spindle/chromosome alignment abnormalities at the 200 ug/kg-day BPA dose level. The soy diet produced an apparent U-shaped dose response. Offspring from the 500 µg/kg-day dose group on the soy diet had a higher abnormal metaphase II rate than that of the combined data for the vehicle and baseline (no vehicle, no BPA dosing) control groups, but no difference was seen for this group as compared with either control group individually.

Cross-reference 6

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Conducted in compliance with OECD Principles of Good Laboratory Practice (GLP), United States Food and Drug Administration GLP Regulations, United States Environmental Protection Agency GLP Standards, the United Kingdom GLP Compliance Programme, and the Japanese GLP Standard. BPA is a constituent of the reaction mass so that its hazard data are relevant for the assessment of the reaction mass.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Male and female ICR mice were dosed by oral gavage with 0, 500, 1000, or 2000 mg/kg BPA. Bone marrow cells were collected at 24 or 48 hours after treatment and were examined microscopically for the presence of micronucleated polychromatic erythrocytes.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc. (Frederick, Maryland, United States)
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: males, 28.9 - 35.5 g; females, 26.2 - 32.2 g
- Assigned to test groups randomly: no, distributed according to body weight
- Fasting period before study: no
- Housing: Mice of the same sex were housed up to five per cage in plastic autoclavable cages maintained on stainless steel racks. Heat-treated hardwood chips were used for bedding.
- Diet: ad libitum, certified laboratory rodent chow (Harlan TEKLAD certified Rodent 7012C) which had been analyzed for environmental contaminants
- Water: ad libitum, tap water
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 68-80
- Humidity (%): 30-70
- Photoperiod: 12 hours light/12 hours dark

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

Mice were assigned to seven experimental groups of five males and five females each according to a computer-generated program which is based on distribution according to body weight. The BPA-vehicle mixture, vehicle alone, or positive control were administered by oral gavage at a constant volume of 20 mL/kg body weight. All mice were weighed immediately prior to dose administration and the dose volume was based on individual body weights.

Duration of treatment / exposure:

Single administration by oral gavage

Frequency of treatment:

Single adminstration

Post exposure period:

24 or 48 hours

Remarks:

Doses / Concentrations:
0, 500, 1000, or 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

10 for vehicle controls and highest test dose (2000 mg/kg); 5 for the low and mid test doses (500 and 1000 mg/kg) and the positive controls

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide at 50 mg/kg

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Bone marrow cells were isolated from femurs and suspensions of cells were spread on glass slides. Two slides were prepared from each mouse. Slides were fixed in methanol, stained with May-Gruenwald-Giemsa, and permanently mounted.

Evaluation criteria:

2000 polychromatic erythrocytes per slide were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated and the proportion of polychromatic to total erythrocytes was recorded per 1000 erythrocytes.

A positive response was induced if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control at any sampling time. If a single treament group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered suspect of unconfirmed positive and a repeat assay recommended. A negative response was determined if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

The criteria for a valid test were described as follows: The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control group. The incidence in the positive control group must be significantly increased relative to the vehicle control group.

Statistics:

The incidence of micronucleated polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distrubution.

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 mortality occurred at any dose level during the course of the study. Lethargy was noted in 2 of 5 male mice and 1 of 5 female mice at 500 mg/kg and in all mice at 1000 and 2000 mg/kg. Piloerection was observed in all mice at 1000 and 2000 mg/kg.

Reductions of 15 to 24% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in male and female dose groups 24 hours after treatment with all doses of BPA. Reductions of 26% and 36% were observed in male and female mice, respectively, 48 hours after treatment with 2000 mg/kg BPA.

The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in BPA-treated groups was not increased relative to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p>0.05).

Conclusions:

Interpretation of results: negative
The authors concluded that under the conditions of the assay, BPA was negative in the micronucleus test using male and female ICR mice.

Executive summary:

Male and female ICR mice were dosed by oral gavage with 0, 500, 1000, or 2000 mg/kg BPA. Bone marrow cells were collected at 24 or 48 hours after treatment and were examined microscopically for the presence of micronucleated polychromatic erythrocytes. No mortality occurred at any dose level during the course of the study. Lethargy was noted in 2 of 5 male mice and 1 of 5 female mice at 500 mg/kg and in all mice at 1000 and 2000 mg/kg. Piloerection was observed in all mice at 1000 and 2000 mg/kg. Reductions of 15 to 24% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in male and female dose groups 24 hours after treatment with all doses of BPA. Reductions of 26% and 36% were observed in male and female mice, respectively, 48 hours after treatment with 2000 mg/kg BPA. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in BPA-treated groups was not increased reltive to their respective vehicle controls in either males or females, regardless of dose level or bone marrow collection time (p>0.05).

Cross-reference 7

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

genetic toxicity in vivo

Remarks:

Type of genotoxicity: other: chromosome aberration; aneugenic effects

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study which meets basic scientific principles. BPA is a constituent of the reaction mass so that its hazard data are relevant for the assessment of the reaction mass.

Qualifier:

no guideline followed

Principles of method if other than guideline:

Experiment 1: Superovulated C57Bl/6 female mice of either four or nine weeks of age were injected with human chorionic gonadotropin (HCG) then immediately treated by gavage with 0.2 or 20 mg/kg BPA. Metaphase II oocytes were harvested 17 hours later and cytogenetically analyzed after C-banding. To study effects of subchronic or chronic BPA exposures, two further groups of female mice, four weeks of age, were either treated with daily oral gavage administrations of 0.04 mg/kg BPA for seven days or exposed for seven weeks to BPA at 0.5 mg/L in drinking water. Two days before the end of the seven-day or seven-week exposure, females were superovulated, then 48 hours later were injected with HCG. The females were mated overnight, and unplugged females were sacrificed to harvest oocytes for cytogenetic analysis and plugged females were used to prepare zygote metaphases to evaluate effects on the second meiotic division. Zygote metaphases were also prepared from plugged females that received a single oral dose of 0.2 mg/kg BPA.

Experiment 2: Meiotic delay in spermatocytes was assessed using 102/ElxC3H/E1 F1 males that were intraperitoneally injected with BrdU in saline and randomly assigned to receive either (1) an oral dose of 0.2 mg/kg BPA for six consecutive days starting on day 8 after BrdU, or (2) a comparable volume of vehicle (corn oil) for the same number of days. Five males from each group were sacrificed on days 21, 22, 23, 24, and 25 after the end of the BPA treatment. Sperm were collected and examined for BrdU incorporation. Twenty more male mice were then treated orally with 0, 0.002, 0.02, or 0.2 mg/kg BPA on six consecutive days. Sperm were collected for multicolor FISH analysis to assess the induction of aneuploidy during the first and second meioitc division.

Experiment 3: Male mice were treated with 0. 0.002, 0.02, or 0.2 mg/kg BPA by gavage on two consecutive days, then 24 hours later, bone marrow smears were prepared for the micronucleus assay.

GLP compliance:

no

Type of assay:

other: micronucleus assay; assessment of meiotic progression

Species:

mouse

Strain:

other: C57Bl/6; 102/E1xC3H/E1

Sex:

male/female

Route of administration:

other: oral gavage; oral: drinking water

Vehicle:

corn oil or water

Additional information on results:

Experiment 1: Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in more than two dyads, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: The proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Experiment 1: negative; Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: negative; the proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: negative; BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Conclusions:

Experiment 1: negative; Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

Experiment 2: negative; the proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

Experiment 3: negative; BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Executive summary:

Experiment 1: Superovulated C57Bl/6 female mice of either four or nine weeks of age were injected with human chorionic gonadotropin (HCG) then immediately treated by gavage with 0.2 or 20 mg/kg BPA. Metaphase II oocytes were harvested 17 hours later and cytogenetically analyzed after C-banding. To study effects of subchronic or chronic BPA exposures, two further groups of female mice, four weeks of age, were either treated with daily oral gavage administrations of 0.04 mg/kg BPA for seven days or exposed for seven weeks to BPA at 0.5 mg/L in drinking water. Two days before the end of the seven-day or seven-week exposure, females were superovulated, then 48 hours later were injected with HCG. The females were mated overnight, and unplugged females were sacrificed to harvest oocytes for cytogenetic analysis and plugged females were used to prepare zygote metaphases to evaluate effects on the second meiotic division. Zygote metaphases were also prepared from plugged females that received a single oral dose of 0.2 mg/kg BPA. Cytogenetic analysis of oocytes revealed no BPA-related effects on aneuploidy with the acute, subchronic, or chronic dosing regimens. The only BPA-related effect observed in oocytes was an increase in the percentage of metaphase II oocytes showing premature centromere separation in the group treated for 7 weeks, as compared to vehicle controls. Cytogenetic analysis of zygotes revealed no BPA-related effects on any parameters examined, such as frequency of zygotes reaching first cleavage division or containing any type of structural or numerical chromosome change.

 

Experiment 2: Meiotic delay in spermatocytes was assessed using 102/ElxC3H/E1 F1 males that were intraperitoneally injected with BrdU in saline and randomly assigned to receive either (1) an oral dose of 0.2 mg/kg BPA for six consecutive days starting on day 8 after BrdU, or (2) a comparable volume of vehicle (corn oil) for the same number of days. Five males from each group were sacrificed on days 21, 22, 23, 24, and 25 after the end of the BPA treatment. Sperm were collected and examined for BrdU incorporation. Twenty more male mice were then treated orally with 0, 0.002, 0.02, or 0.2 mg/kg BPA on six consecutive days. Sperm were collected for multicolor FISH analysis to assess the induction of aneuploidy during the first and second meioitc division. The proportion of X and Y chromosome-bearing sperm did not differ from the ratio of 1:1 and there was no increase in frequency of hyperhaploid or diploid sperm with any dose of BPA.

 

Experiment 3: Male mice were treated with 0. 0.002, 0.02, or 0.2 mg/kg BPA by gavage on two consecutive days, then 24 hours later, bonem arrow smears were prepared for the micronucleus assay. BPA did not induce micronuclei in bone marrow erythrocytes at any dose.

Cross-reference 8

Reason / purpose for cross-reference:

read-across source

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study is comparable to OECD guideline 474 concerning the micronucleus test without relevant restriction and the experiments on hypothermia in mice are according to generally accepted standard methods. Study compliant with Good Laboratory Practice Standards. Minor restrictions: individual test results in the micronucleus test not reported; 200 mg/kg bw not tested in the micronucleus test but slightly reduced body temperature at this dose [threshold]. Phenol is a constituent of the reaction mass so that phenol hazard data are applied in the hazard assessment.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

The micronucleus test in mice was combined with investigations on hypothermia induced by phenol at high dose levels to study a possible relationship.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- outbred CD-1(1CR)BR
- Source: Charles River Laboratories (Portage, MI, USA)
- Age at study initiation: 9 weeks
- Weight at study initiation: no data
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: individually
- Diet (e.g. ad libitum): certified rodent diet, analysed for contaminations
- Water (e.g. ad libitum): municipal drinking water, analysed for contaminations
- Acclimation period: 7 days
- Randomisation: by computer program, based on body weight, uniform group mean weight

ENVIRONMENTAL CONDITIONS
- adequate environmental conditions (temperature 21-23°C, rel. humidity 40-70%, and photocycle 12/12h)

Route of administration:

intraperitoneal

Vehicle:

- Vehicle: phosphate buffered saline
- Justification for choice of solvent/vehicle: aqueous solution recommended (see OECD 474)
- Concentration of test material in vehicle: 0, 0.3, 1.0, 3.0% at 0, 30, 100, 300 mg/kg bw in Micronucleus (MN) assay
- Application volume: 10 ml/kg bw in all experiments

Details on exposure:

1) Hypothermia Experiment: the maximum tolerated dose (MTD) for a single ip injection & potential for phenol to induce hypothermia were studied.
2) Thereafter the micronucleus assay followed to study the relationship between phenol induced hypothermia and the induction of micronuclei (MN).
3) In a further independent experiment the kinetochore status of MN as a measure of spindle disturbances was measured to study a possible mechanism (impairment of microtubule assembly during mitosis).

1) Hypothermia
i.p. vehicle control (PBS) or 50, 100, 150, 200, 300, 400, or 500 mg/kg bw; relative body temperature (BT) monitored sc using programmable transponders (also served for identification). BT determined prior to dosing and at 5, 30, 60, and 90 min and 2, 3, 4, 5, 6, 24, and 48 h after dosing; clinical signs of toxicity evaluated in each mouse at the same time points as BT.

2) MN assay: single ip injection of 0, 30, 100, or 300 mg/kg bw; positive control: single 120 mg/kg bw dose of cyclophosphamide by oral gavage; BT and clinical observations of animals in all dose groups monitored using above desribed procedure, just prior to dosing and at 2, 5, 24, and 48 h after dosing; mice killed either 24 or 48 h after dosing; bone marrow sample collected from femurs.

3) Kinetochor experiment
0 or 300 mg/kg bw (the only dose to cause an increased frequency in MN in the initial MN assay) plus positive control vinblastin; mice killed 24 h after dosing and bone marrow sample collected.

Duration of treatment / exposure:

All experiments: single i.p. application (except cyclophosphamide, single gavage).

Frequency of treatment:

once

Post exposure period:

Hypothermia experiment: 48 h
MN assay: 24 or 48 h (positive control only 24 h)
Kinetochor assay: 24 h

Remarks:

Doses / Concentrations:
0, 50, 100, 150, 200, 300, 400, or 500 mg/kg bw
Basis:
other: actual dose in hypothermia experiment (no positive control)

Remarks:

Doses / Concentrations:
0, 30, 100, or 300 mg/kg bw
Basis:
other: actual dose in MN assay plus positive control cyclophosphamide

Remarks:

Doses / Concentrations:
0, 300 mg/kg bw
Basis:
other: actual dose in kinetochor experiments plus positive control vinblastin

No. of animals per sex per dose:

Hypothermia assay: 4 m & 4 f per dose
MN assay: 6 m & 6 f per dose per survival time
Kinetochor test: 6 m per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

1) no positive control in hypothermia test
2) MN assay: cyclophosphamide monohydrate
- Route of administration: oral, gavage
- Doses / concentrations: 120 mg/kg bw in 10 ml/kg bw
3) kinetochore-positive MN: Vinblastin (i.p. 4 mg/kg bw in 10 ml/kg bw)

Tissues and cell types examined:

1) Hypothermia test: BT and clinical signs
2) MN assay: BT and clinical signs plus MN in bone marrow erythrocytes after sacrifice.
3) Kinetochor test: MN with kinetochor in bone marrow erythrocytes

Details of tissue and slide preparation:

MN assay: Bone marrow samples prepared for light microscopy; cell pellets resuspended in a drop of serum and film prepared on a slide; slides dried prior to staining with Wright-Giemsa. Slides coded & scored on a blind basis; 2000 polychromatic erythrocytes examined per mouse and number of micronucleated
polychromatic erythrocytes (MN-PCE) recorded; the ratio of PCE to normochromatic erythrocytes (NCE) in the bone marrow was determined in ca. 200 erythrocytes from each animal; ratio expressed as percentages: (PCE x 100/PCE + NCE).

Kinetochor test: bone marrow cells dried on a slide followed by kinetochore staining consisted of attaching and stacking a series of antibodies to the kinetochores of chromosomes; thereafter slides stained with propidium iodide (1–5 µl) for 1–2 min, rinsed and mounted with 100–200 µg/ml of phenylenediamine in glycerol. Microscopy on a blind basis (slides coded); evaluation of MN with kinetochores: up to 20 MN/aimal were analyzed for the occurrence of kinetochores.

Evaluation criteria:

No data but clear positive results in the MN assay. Significance shown by statistical analysis (see below).

Statistics:

The raw data of MN-PCE transformed by adding one to each count; transformed MN-PCE data and the data on percent PCE analyzed separately by a three-way analysis of variance (Winer, 1971). Thereafter the two-way interactions reviewed for significance. The data then analyzed by one, two, or three-way analysis looking only at main effects. Pairwise comparisons of treated versus control groups if the dose effect was significant, by Dunnett t-test, one sided (upper) for MN-PCE and two sided for the percent PCE (Winer, 1971). Linear dose-related trend tests performed if any of the pairwise comparisons yield significant differences. Kinetochore-positive MN-PCEs compared using Fisher exact test. The alpha level at which all the test data were conducted was 0.05.

Sex:

male/female

Genotoxicity:

positive

Remarks:

at high dose level showing also hypothermia

Toxicity:

yes

Remarks:

clinical signs plus hypothermia

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1) Hypothermia test
- mortality: at >= 400 mg/kg bw mice died within 24 h after application; at 300 mg/kg bw 1 male and 1 female died within the post exposure observation period; no mortality in other dose groups.
- clinical signs: >= 100 mg/kg bw twitching and tremors, >= 200 mg/kg bw decreased activity in females, >= 300 decreased activity in males;
surviving mice appeared normal approximately 1 h after application and exhibited no further clinical signs.
- body temperature (BT) revealed dose dependent effects; no effect at 50 mg/kg bw; slight decrease (1-2°C) 30 min after application at 100 & 150 mg/kg bw in males and at 100 mg/kg bw in females; at 150 mg/kg bw decrease in females of max. 3°C for 2-3 h; at 200 mg/kg bw max. 3-4°C decrease for 3-4 h in males and females; at 300 mg/kg bw mean BTs decreased to approximately 32°C after 30 min. with mean BT as low as 28°C at 5 h after dosing in both sexes; BT remained depressed 4–5°C even at termination (48h); at >= 400 mg/kg bw the hypothermia was aggravated (but animals died, see above).
- body weight: no effects

2) Mikronucleus test
- no treatment related mortality
- at 300 mg/kg bw one third of the male mice and one half of the female mice showed clinical signs (twitching/tremors) within minutes after dosing and persisted for approximately 1 h; at 100 mg/kg bw twitching/tremors persisted only for several minutes; no treatment-related effects at 30 mg/kg bw.
- 24 and 48 h after application the body temperature was not affected in positive control and at doses lower than 300 mg/kg bw; at the high dose of 300 mg/kg bw the effects were consistent with results in the hypothermia prestudy; 24 hours after dosing mean BTs decreased to 31.5°C in both sexes, 48 h after treatment the degree of BT decrement was 7°C (28.6°C) in male mice and 6°C (30.1°C) in female mice.
- at 300 mg/kg significant increases in the frequency of MN-PCE at 24 and 48 h after dosing; no effects at lower dose levels (see Table)
- the decrease in PCE/NCE ratio (see Table above) was obvious at 300 mg/kg bw and indicated significant effect of the treatment on erythropoiesis.

3) Kinetochor experiment
significant increase in the proportion of kinetochore-positive (K+) MN observed at 300 mg/kg bw compared to controls; The proportion of (K+) MN induced by the positive control vinblastin > observed for phenol (78% versus 13%). Large proportion of MN after phenol treatment not K+ indicating other mechanisms.

MN-PCE Frequencies and % PCE after i.p. application of phenol in mice

Dose in mg/kg bw	Decrease (in °C) of body temperature after 48 h compared to predosing	MN-PCE per 1000 PCE	% PCE of total erythrocytes	MN-PCE per 1000 PCE	% PCE of total erythrocytes
		Harvest time 24 h		Harvest time 48 h
	Males
0	-1.9	2.1+-1.8	64.2	1.1+-0.4	63.8
30	-0.1	4.3+-1.8	52.0	1.3+-1.0	65.9
100	-0.3	3.1+-1.5	50.3	1.5+-1.4	65.1
300	-7.1	10.8+-8.5*	50.3	18.3+-1.8*	37.8
Pos. control	-0.3	79.9+-20.1*	37.2	NE	NE
	Females
0	-0.5	2.5+-2.0	68.0	2.4+-1.5	64.0
30	+0.1	1.1+-1.1	57.3	1.0+-0.8	65.1
100	-0.1	2.3+-0.8	60.3	1.1+-0.5	63.6
300	-6.2	11.3+-9.3*	42.3	17.8+-14.3*	33.3
Pos. control	+0.8	88.0+-28.7*	58.8	NE	NE
*: significant (alpha =0.05); NE: not examined

Conclusions:

Interpretation of results: threshold dependent chromosome mutagenic activity
MN formation exhibited a dose threshold correlated with phenol-induced hypothermia

Executive summary:

The study is comparable to OECD guideline 474. The additional experiments on hypothermia and aneugenic effects are according to generally accepted standard methods. The study is in compliance with Good Laboratory Practice Standards.

In a preliminary study the dose dependent effects of phenol on body temperature and clinical signs & survival after a single i.p. injection was studied in male and female CD-1 mice for 48 h (n=4 per dose per sex; 0, 50, 100, 150, 200, 300, 400, or 500 mg/kg bw). At >= 400 mg/kg bw mice died within 24 h after application. Clinical signs occurred at >=100 mg/kg bw but survivors appeared normal approximately 1 h after application. However, at 300 mg/kg bw (or above) significant and prolonged hypothermia in male and female mice (up to 7°C decrease) was detected.

In the Micronucleus (MN) assay males and females were killed 24 and 48 h after a single i.p. application (n=6 per dose per sex; i.p. 0, 30, 100, 300 mg/kg bw) and the incidence of MN in bone marrow was measured. Prolonged hyperthermia was found only in the high dose group as well as a significant increase in micronuclei. No clastogenic effects were reported at lower dose levels. These results suggested a threshold mechanism for the induction of MN by phenol treatment in mice via prolonged physiologic hypothermia.

In additional experiments a significant increase in kinetochore-positive MN was observed at 300 mg/kg bw, but the response was considerably less than that the known spindle poison vinblastin indicating that the interruption of the cell spindle apparatus appeared to play only a minor role in MN formation.

Conclusion: Micronucleus formation exhibited a dose threshold correlated with phenol-induced hypothermia.

Materials and methods

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 4,4'-isopropylidenediphenol and phenol

EC Number:

904-653-0

IUPAC Name:

Reaction mass of 4,4'-isopropylidenediphenol and phenol

Results and discussion

Test results

Test results 1

Sex:

male/female

Genotoxicity:

negative

Remarks:

(BPA)

Toxicity:

no effects

Test results 2

Sex:

male/female

Genotoxicity:

positive

Remarks:

(Phenol)

Toxicity:

yes

Remarks on result:

other: weak positive results in micronucleus tests were found at dose levels inducing severe signs of intoxication; significant increase in micronuclei was found only in the high dose groups with prolonged hypothermia

Applicant's summary and conclusion

Conclusions:

No experimental data are available for the target substance Reaction mass of phenol and 4,4’-isopropylidenediphenol. However, based on the general principles of mixture toxicology, data on the main constituents of this multi-constituent substance are used as surrogate. Based on the availabel data, it does not appear that Bisphenol A has significant mutagenic or genotoxic potential in vivo. However, teh constituent Phenol is classified as Muta 2, but the results from in vivo test systems suggested a possible threshold mechanism above 100 mg/kg bw/d for the induction of micronuclei via prolonged hypothermia.