4-hydroxybenzophenone

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 Dec 2022 - 20 Jan 2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzophenone

EC Number:

214-507-1

EC Name:

4-hydroxybenzophenone

Cas Number:

1137-42-4

Molecular formula:

C13H10O2

IUPAC Name:

4-benzoylphenol

Test material form:

solid: particulate/powder

Details on test material:

Identification: 4-hydroxy-benzophenone
Molecular formula: C13H10O2
Molecular weight: 198.22
CAS Number: 1137-42-4
Description: White crystal
Purity: 99.72 %

Specific details on test material used for the study:

Physical Description: White powder
Purity/Composition: 99.5%
Storage Conditions: At room temperature protected from light
CAS number: 1137-42-4
Chemical name: 4-hydroxybenzophenone

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on species / strain selection:

The Wistar WI (Han) rat was used as the test system because it is a readily available rodent species, which is commonly used for this purpose, with documented susceptibility to a wide range of toxic materials.

Sex:

male

Details on test animals or test system and environmental conditions:

Species: Rat
Strain: Crl: WI(Han)
Condition: Outbred, SPF-Quality
Source: Charles River Deutschland, Sulzfeld, Germany.
Number of Males: 3 (dose range finding), 26 (main study)
Number of Females: 3 (dose range finding)
Age at the Initiation of Dosing: 6 weeks

Caging: Polycarbonate cages (Makrolon type IV or 2000P Tecniplast) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Up to 5 animals of the same sex and same dosing group were housed together.

Cage Identification: Colour-coded cage card indicating Test Facility Study No., group, animal number(s).

Animal Enrichment - Animals were socially housed for psychological/environmental enrichment and were provided with materials such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.

ENVIRONMENTAL CONDITIONS
-The targeted conditions for animal room environment will be as follows:
Temperature: 20 to 24°C
Humidity: 40 to 70%
Light Cycle: 12 hours light and 12 hours dark (except during designated procedures)
Ventilation: Ten or more air changes per hour
The actual daily mean temperature during the study period was 20.7 to 22.7°C with an actual daily mean relative humidity of 48 to 55%.

Food
Diet: SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany
Type: Pellets
Frequency: Ad libitum, except during designated procedures.
Analysis: Results of analysis for nutritional components and environmental contaminants were provided by the supplier and are on file at the Test Facility. It is considered that there were no known contaminants in the feed that would interfere with the objectives of the study.

Water
Type: Municipal tap water.
Frequency/Ration: Freely available to each animal via water bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

The vehicle for this test material was propylene glycol (PG).

Details on exposure:

A solubility test was performed based on visual assessment. The test material was suspended in propylene glycol (Merck, Darmstadt, Germany). The density of vehicle is 1.036 g/mL. Test material concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension.
This resulted in white suspensions for all formulations. Test material concentrations were dosed within 4 hours after preparation.
Any residual volumes were discarded.

Duration of treatment / exposure:

two consecutive days

Frequency of treatment:

Once daily on two consecutive days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control slides taken from male animals previously dosed with positive control (cyclophosphamide, 19 mg/kg bw at 10 mL/kg dosed orally 48 hours prior to sampling), as part of Test Facility Study No. 20337827, were added to the study slides for evaluation as scoring controls.

Examinations

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

Bone marrow was sampled 48 hours after the first dosing. 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. The bone was flushed with approximately 4 mL of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 216 g for 5 min.
The supernatant was removed with a Pasteur pipette. Approximately 500 µl 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 slide. 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. At least two slides were prepared per animal.
The slides were automatically stained using the "Wright-stain-procedure" in a HEMA-tek slide stainer (Hematek 3000, Siemens Healthcare, Den Haag, The Netherlands). This staining is based on Giemsa. The dry slides were automatically mounted with a coverslip with an automated coverslipper (ClearVue Coverslipper, Thermo Fisher Scientific, Breda, The Netherlands).

Evaluation criteria:

To prevent bias, all slides were randomly coded before examination. An adhesive label with 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 4000 polychromatic erythrocytes (with a maximum deviation of 5%). The proportion of immature erythrocytes was determined by counting and differentiating at least the first 500 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. Parts on the slides that contained mast cells that might interfere with the scoring of micronucleated polychromatic erythrocytes were not used for scoring.
A micronucleus test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control material induces a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

Statistics:

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data.
A test material is considered positive in the micronucleus test if all of the following criteria are met:
a) At least one of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.
A test material is considered negative in the micronucleus test if:
a) None of the treatment groups exhibits a statistically significant (one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are within the 95% control limits of the negative historical control data range.
Due to inhomogeneous variances the Welch t test with Bonferonni-Holm Adjustment was applied.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In the dose-range finding test, three male and three female animals dosed with 2000 mg test material per kilogram body weight showed little related clinical signs or mortality.

RESULTS OF DEFINITIVE STUDY
Based on the results of the dose-range finding study dose levels of 500, 1000 and 2000 mg/kg body weight were selected as appropriate doses for the micronucleus main test. Since there were no differences between sexes in toxicity only male animals were used in the main study, Five male and five female animals were used in each treatment group.
The animals of the groups treated with test material and the animals of the negative group showed no treatment related clinical signs of toxicity or mortality.

Micronucleated Polychromatic Erythrocytes
The mean number of micronucleated polychromatic erythrocytes scored in test material treated groups were compared with the corresponding solvent control group. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of the test material treated animals compared to the vehicle treated animals. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the within the 95% control limits of the distribution of the historical negative control database.
Cyclophosphamide, the positive control material, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. In addition, the number of micronucleated polychromatic erythrocytes found in the positive control animals was within the 95% control limits of the distribution of the historical positive control database. Hence, all criteria for an acceptable assay were met.

Proportion of Immature Erythrocytes
The animals of the groups, which were treated with the test material and the positive control slides showed no decrease in the proportion of immature erythrocytes, which indicated a lack of toxic effects of this test material on the erythropoiesis.

Bioanalysis
Blood was sampled 1, 2, 4, 6 and 24 h after the second dose of TK animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

Any other information on results incl. tables

Table 1 - Mortality and Toxic Signs after Treatment with the Test Material in the Dose-range Finding Study

Group

Sex

Toxic signs*

Animal

Dose

Number

mg/kg

day 1 post-dose within

day 2 pre-dose

day 2 post-dose within

day 3

2.5 hrs

2.5 hrs

1

Male

101

2000

B

B

N

1

Female

102

2000

B

B

N

1

Male

103

2000

B

B

N

B

1

Male

104

2000

B

N

N

B

1

Female

105

2000

X

J X

J X

B

1

Female

106

2000

B

J

J

B

* Legend 'Mortality and toxic signs':
B = showed no abnormalities; J = hunched posture; N = rough coat; X = rattling breathing

 

Table 2 - Mean Body Weight

Sex: Male		Day(s) Relative to Start Date
		1	2	3
0	Mean	176.2	183.6	188.6
mg/kg/day	SD	7.6	9.2	7.4
Group 1	N	5	5	5
500	Mean	175.6	181.4	186.4
mg/kg/day	SD	7.6	9.1	10
Group 2	N	5	5	5
	%Diff	-0.3	-1.2	-1.2
1000	Mean	175.8	183.2	184
mg/kg/day	SD	4.9	5.4	5.9
Group 3	N	5	5	5
	%Diff	-0.2	-0.2	-2.4
2000	Mean	176.4	185.2	189
mg/kg/day	SD	5.3	7.2	6.8
Group 4	N	5	5	5
	%Diff	0.1	0.9	0.2

Bodyweight (g)

 

Table 3 - Mean Number of Micronucleated Polychromatic Erythrocytes and Proportion of Immature Erythrocytes

Group	Treatment	Number of Animals	Dose	Number of micronucleated polychromatic erythrocytes			Proportion of Immature Erythrocytes
			(mg/kg body weight)	(mean ± S.D.) ^(1)			(mean ± S.D.) ^(2)
	MALES
1	Vehicle Control	5	0	2.8	±	2.2	0.49	±	0.06
2	Test Material	5	500	4.2	±	0.8	0.54	±	0.07
3	Test Material	5	1000	4	±	1.9	0.6	±	0.07
4	Test Material	5	2000	3.8	±	0.8	0.52	±	0.06
5	CP	3	19	20	±	2	0.41	±	0.08

Vehicle control = PG
CP = Cyclophosphamide.
(1) At least 4000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(2) The proportion was determined from at least the first 500 erythrocytes counted.
(3) Significantly different from corresponding control group (Students t test, P < 0.001).

 

Table 4 - Individual Data

		Number of polychromatic erythrocytes (1)	Number of normochromatic erythrocytes^(1)	Proportion of Immature Erythrocytes ^(1)	Number of micronucleated polychromatic erythrocytes	Number of polychromatic erythrocytes scored for micronuclei
Group	Animal number
1	1	214	286	0.43	1	4000
1	2	253	247	0.51	2	4000
1	3	291	209	0.58	4	4000
1	4	216	284	0.43	1	4000
1	5	258	242	0.52	6	4000
2	6	240	260	0.48	5	4000
2	7	301	199	0.6	4	4000
2	8	245	255	0.49	3	4000
2	9	314	186	0.63	5	4000
2	10	249	251	0.5	4	4000
3	11	289	211	0.58	2	4000
3	12	284	216	0.57	6	4000
3	13	341	159	0.68	2	4000
3	14	332	168	0.66	5	4000
3	15	264	236	0.53	5	4000
4	16	278	222	0.56	3	4000
4	17	251	249	0.5	3	4000
4	18	233	267	0.47	4	4000
4	19	239	261	0.48	4	4000
4	20	303	197	0.61	5	4000
5	21	168	332	0.34	22	4000
5	22	243	257	0.49	18	4000
5	23	203	297	0.41	20	4000

(1) The proportion was determined from the first 500 erythrocytes counted.

 

Table 5 - Historical Negative Control Data for Micronucleus Studies

	Male
Mean Number of Micronucleated cells per 4000 cells	3.7
SD	1.6
n	70
Lower Control Limit (95% Control Limits)	1
Upper Control Limit (95% Control Limits)	7

SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between November 2019 and November 2022.

 

Table 6 - Historical Positive Control Data for Micronucleus Studies

	Male
Mean Number of Micronucleated cells per 4000 cells	33.7
		n	65
SD	22.7
Lower Control Limit (95% Control Limits)	-11
Upper Control Limit (95% Control Limits)	78

SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between November 2019 and November 2022.

 

Applicant's summary and conclusion

Conclusions:

4-hydroxybenzophenone CAS# 1137-42-4 is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats 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:

The objective of the study was to obtain information on the clastogenicity and aneugenicity of 4-hydroxybenzophenone CAS# 1137-42-4 when administered to rats at a maximum required acute dose, by measuring the increase in the number of micronucleated polychromatic erythrocytes per 4000 polychromatic erythrocytes in rat bone marrow.

Based on the results of the dose-range finding study test a concentration of 2000 mg/kg/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline). Since there were no substantial differences in toxicity between sexes only males were used in the main study.

The formulations were homogeneous (i.e. coefficient of variation ≤ 10%). In the main study male animals were dosed twice by oral gavage with vehicle or with 500, 1000 and 2000 mg test material per kg body weight. For the positive control, slides from animals dosed once by oral gavage with 19 mg cyclophosphamide (CP) per kg body weight were used. In total 4 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal treated with the test material or control animals receiving vehicle or cyclophosphamide.

 

Bone marrow was sampled 48 hours after the first dosing. No biological relevant increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with the test material compared to the vehicle treated animals.

The groups that were treated with the test material and positive control slides showed no decrease in the proportion of immature erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test material on erythropoiesis.

Blood was sampled 1, 2, 4, 6 and 24 h after the second dose of TK animals dosed with the vehicle and the highest concentration of the test material. Vehicle dosed animals showed levels below the lower limit of quantification in the plasma. All test material dosed animals showed increased levels of the test material in the plasma, confirming systemic exposure.

In conclusion, 4-hydroxybenzophenone CAS# 1137-42-4 is not clastogenic or aneugenic in the bone marrow micronucleus test of male rats 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.