Carbonic acid, diphenyl ester, reaction products with bisphenol A

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

key study

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].

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

2007

Reference 2

Reference Type:

publication

Title:

Potential for Phenol to disrupt the spindle apparatus as evaluated in the mouse bone marrow micronucleus test (MNT)

Author:

Spencer PJ, Linscombe VA, Grundy JG, Gollapudi BB, Waechter JM, Dimond SS

Year:

2004

Bibliographic source:

Toxicologist 78: 29 (abstract)

Reference 3

Reference Type:

publication

Title:

Induction of micronuclei by phenol in mouse bone marrow is associated with treatment-induced hypopthermia

Author:

Spencer PJ, Hammond TA, Waechter JM, Gingell R, Dimond SS, Dunn BJ, Butala JH

Year:

2002

Bibliographic source:

Toxicol 66: 72

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Phenol

EC Number:

203-632-7

EC Name:

Phenol

Cas Number:

108-95-2

Molecular formula:

C6H6O

IUPAC Name:

phenol

Details on test material:

- Analytical purity: 100% by GC/TCD area%
- Impurities (identity and concentrations): no
- Lot/batch No.: 112769
- Stability under test conditions: concentrations in vehicle were analysed in Hypothermia Experiment: 97-103% of the targeted values (HPLC/UV technique and external standard quantification); similar results in other experiments
- Source: Harrell Industries, Rock Hill, South Carolina

Test animals

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)

Administration / exposure

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 described 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

Doses / concentrations

Doses / concentrations 1

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)

Doses / concentrations 2

Remarks:

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

Doses / concentrations 3

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)

Examinations

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/animal 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.

Results and discussion

Test results

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) Micronucleus 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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): other: 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.