Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26-Nov-2013 to 23-Jan-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: lumps
Details on test material:
- Name of test material (as cited in study report): 4, 4’-Isopropylidenediphenol propoxylated (BPA+2PO)
- Substance type: White lumps
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark


Test animals

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

Administration / exposure

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.
Doses / concentrations
Remarks:
Doses / Concentrations:

Basis:
nominal conc.
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

Examinations

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

Results and discussion

Test results
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
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.