4,4'-cyclohexylidenedi-o-cresol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-cyclohexylidenedi-o-cresol

EC Number:

219-110-7

EC Name:

4,4'-cyclohexylidenedi-o-cresol

Cas Number:

2362-14-3

Molecular formula:

C20-H24-O2

IUPAC Name:

4-[1-(4-hydroxy-3-methylphenyl)cyclohexyl]-2-methylphenol

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Dimethyl cyclohexyl bisphenol (DMBPC; CAS No. 2362-14-3)
- Synonyms: Dimethyl bisphenolcyclohexane; 4,4’-cyclohexylidene di-o-cresol; 1,1-Bis(4-hydroxy-3-ethyl)cyclohexane; Bis-OC-Z
- Appearance: fine white powder

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

The ICR mice were obtained from the supplier and were approximately 6-8 weeks of age at study initiation, weighing 24.5 to 31.4 g (males) and 23.6 to 28.3 g (females). Up to five mice of the same sex were group housed in polycarbonate cages with heat-treated hardwood chips for bedding. The controlled environmental parameters were 72 ± 3 °F, 50 ± 20 % relative humidity and a 12-hour light/dark cycle. The mice had free access to certified rodent chow and tap water.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Corn oil (from ICN Biomedicals)

Details on exposure:

A workable suspension of the test substance in corn oil at 100 mg/mL – the maximum concentration tested in the study.

In the pilot study, DMBPC was administered to 2 male mice each at 1, 10, 100, or 1000 mg/kg and to 5 male and 5 and female mice at 2000 mg/kg. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.

In the toxicity assay, 5 male and 5 female mice each were dosed with 200, 400, 600 or 800 mg test article/kg body weight. Mice were observed after dose administration and daily thereafter for 3 days for clinical signs of toxicity. Body weights were recorded prior to dose administration and 1 and 3 days after dose administration.

Mortality was observed in 5/5 male mice and 3/5 female mice at 400 mg/kg and in all males and females at 600 and 800 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 200, 400, 600 and 800 mg/kg. In addition, ataxia was seen in females at 800 mg/kg. The high dose for the micronucleus test was set at 250 mg/kg, which was estimated to be the maximum tolerated dose.

For the definitive micronucleus assay, mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Additional replacement animals (5 animals/sex) were included in the high dose group, 250 mg/kg, to ensure that the availability of 5 animals/sex for micronucleus analysis. DMBPC vehicle mixture, the vehicle alone or the positive control was administered by a single IP injection at a dose volume of 20 mL/kg body weight. All mice in the experimental and control groups were weighed immediately before dose administration and the dose volume was based on individual body weight. Mice were observed after dose administration for clinical signs of toxicity.

Frequency of treatment:

Single treatment

Post exposure period:

3 days

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations: 1, 10, 100, 1000 or 2000 mg/kg body weight (nominal) - Pilot assay

Doses / concentrations 2

Remarks:

Doses / Concentrations: 200, 400, 600 or 800 mg/kg body weight (nominal) - Toxicity assay

Doses / concentrations 3

Remarks:

Doses / Concentrations: 62.5, 125 or 250 mg/kg body weight (nominal) - Definitive assay

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide monohydrate (CP, CAS No. 6055-19-2); 50 mg/kg

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

At the scheduled sacrifice times, five mice per sex per dose were sacrificed by CO₂ asphyxiation. Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 mL foetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes, and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were re-suspended by aspiration with a capillary pipette and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, stained with May Gruenwald Giemsa and permanently mounted.

Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analysed for the presence of micronuclei. Polychromatic erythrocytes are young, immature red blood cells that stain bluish while normochromatic erythrocytes or normocytes are mature red blood cells that stain pink. Micronuclei are round, darkly-staining nuclear fragments with a sharp contour and diameters usually from 1/20 to 1/5 of an erythrocyte. Micronuclei can occur in both PCEs (MPCEs) and NCEs (MNCEs).
To control for bias, slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes (PCEs/ECs ratio).

Evaluation criteria:

To quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and dose group.
As a guide to interpretation of the data, a test substance was considered to induce a positive response if a dose-responsive increase in micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p less than or equal to 0.05, Kastenbaum Bowman Tables) at any sampling time. If a single treatment group was significantly elevated at one sacrifice time with no evidence of a dose-response, the assay was considered a suspect or unconfirmed positive and a repeat assay recommended. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent vehicle control was observed at any sampling time.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and dose group. Statistical significance was determined using the Kastenbaum Bowman tables which are based on the binomial distribution. All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

In the pilot study, DMBPC was administered to two male mice each at 1, 10, 100, and 1000 mg/kg and to five male and five and female mice at 2000 mg/kg. Mortality was observed in 2/2 male mice at 1000 mg/kg and in 5/5 male mice and 5/5 female mice at 2000 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in all male mice at 1, 10, 100 and 1000 mg/kg and in all male and female mice at 2000 mg/kg.

In the toxicity assay, five male and five female mice each were dosed with 200, 400, 600 or 800 mg test article/kg body weight. Mortality was observed in 5/5 male mice and 3/5 female mice at 400 mg/kg and in all males and females at 600 and 800 mg/kg. Clinical signs following dose administration included: lethargy and piloerection in males and females at 400, 600 and 800 mg/kg. In addition, ataxia was seen in 4/5 females at 800 mg/kg. No clinical signs were observed in any of the animals in the 200 mg/kg dose group. The high dose for the micronucleus test was set at 250 mg/kg, which was estimated to be the maximum tolerated dose.

In the definitive micronucleus test, male and female mice were dosed with DMBPC by a single intraperitoneal injection of 62.5, 125 or 250 mg/kg as well as with the vehicle (corn oil) and positive control (CP) articles. The vehicle and dosing formulations were administered in a total volume of 20 mL/kg body weight. No mortality occurred at any dose level during the course of the micronucleus study. Clinical signs following dose administration included: lethargy and piloerection in all male mice and all female mice at 250 mg/kg. All other animals treated with the test or control articles appeared normal following dose administration.

Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight to moderate reductions of 12 to 33 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time (p greater than 0.05, Kastenbaum-Bowman Tables). CP induced a statistically significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than or equal to 0.05, Kastenbaum-Bowman Tables).

In this study, all criteria for a valid test were met as specified in the protocol. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p less than or equal to 0.05, Kastenbaum-Bowman Tables). The negative and positive controls were consistent with the historical control data, indicating that there was no problem with the test system or the quality of the test.

Any other information on results incl. tables

Summary of Bone Marrow Micronucleus Analysis Following a Single Dose of DMBPC (CAS No.2362-14-3) in ICR Mice

 

Treatment	Sex	Time (hr)	Number of Mice	PCE/Total Erythrocytes (Mean ± SD)	Change from Control (%)	Micronucleated Polychromatic Erythrocytes
						Number per 1000 PCEs (Mean ± SD)	Number per PCEs Scored
Corn oil (20 mL/kg)	M	24	5	0.469 ± 0.09	---	0.4 ± 0.42	4 / 10000
	F	24	5	0.390 ± 0.09	---	0.4 ± 0.42	4 / 10000
DMBPC
62.5 mg/kg	M	24	5	0.409 ± 0.04	-13	0.3 ± 0.27	3 / 10000
	F	24	5	0.397 ± 0.08	2	0.4 ± 0.22	4 / 10000
125 mg/kg	M	24	5	0.347 ± 0.02	-26	0.4 ± 0.22	4 / 10000
	F	24	5	0.344 ± 0.07	-12	0.5 ± 0.35	5 / 10000
250 mg/kg	M	24	5	0.312 ± 0.03	-33	0.4 ± 0.22	4 / 10000
	F	24	5	0.321 ± 0.07	-18	0.5 ± 0.00	5 / 10000
CP
50 mg/kg	M	24	5	0.317 ± 0.02	-32	26.0 ± 3.02	*260 / 10000
	F	24	5	0.289 ± 0.04	-26	27.4 ± 4.72	*274 / 10000
Corn oil (20 mL/kg)	M	48	5	0.415 ± 0.05	---	0.1 ± 0.22	1 / 10000
	F	48	5	0.392 ± 0.04	---	0.5 ± 0.00	5 / 10000
DMBPC
250 mg/kg	M	48	5	0.351 ± 0.09	-15	0.3 ± 0.27	3 / 10000
	F	48	5	0.327 ± 0.01	-17	0.2 ± 0.27	2 / 10000
*Statistically significant, p less than or equal to 0.05 (Kastenbaum‑Bowman Tables)

 

 

 

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours Following a Single Dose of DMBPC (CAS No. 2362 -14 -3)in ICR Mice

Treatment (mg/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil (20 mL/kg)	M	101	0.323	1 / 2000
		102	0.455	2 / 2000
		103	0.507	1 / 2000
		104	0.548	0 / 2000
		105	0.513	0 / 2000
	F	106	0.346	0 / 2000
		107	0.436	1 / 2000
		108	0.325	2 / 2000
		109	0.528	0 / 2000
		110	0.314	1 / 2000
DMBPC
62.5	M	111	0.413	0 / 2000
		112	0.351	1 / 2000
		113	0.454	0 / 2000
		114	0.421	1 / 2000
		115	0.408	1 / 2000
	F	116	0.467	1 / 2000
		117	0.420	1 / 2000
		118	0.266	0 / 2000
		119	0.410	1 / 2000
		120	0.423	1 / 2000
125	M	121	0.371	1 / 2000
		122	0.326	0 / 2000
		123	0.364	1 / 2000
		124	0.317	1 / 2000
		125	0.358	1 / 2000
	F	126	0.324	1 / 2000
		127	0.276	1 / 2000
		128	0.466	0 / 2000
		129	0.312	2 / 2000
		130	0.341	1 / 2000
250	M	131	0.284	1 / 2000
		132	0.361	1 / 2000
		133	0.323	0 / 2000
		134	0.277	1 / 2000
		135	0.315	1 / 2000
	F	136	0.341	1 / 2000
		137	0.233	1 / 2000
		138	0.416	1 / 2000
		139	0.325	1 / 2000
		140	0.288	1 / 2000
CP 50	M	141	0.340	48 / 2000
		142	0.328	50 / 2000
		143	0.324	55 / 2000
		144	0.317	61 / 2000
		145	0.277	46 / 2000
	F	146	0.230	48 / 2000
		147	0.269	46 / 2000
		148	0.300	70 / 2000
		149	0.321	54 / 2000
		150	0.324	56 / 2000

 

 

 

Induction of Micronucleated Polychromatic Erythrocytes inBone Marrow Cells Collected 48 Hours Following a Single Dose of DMBPC

(CAS No. 2362 -14 -3) in ICR Mice

Treatment (mg/kg)	Sex	Animal Number	PCE/Total Erythrocytes	Micronucleated PCE (Number/PCE scored)
Corn oil (20 mL/kg)	M	151	0.405	0 / 2000
		152	0.430	0 / 2000
		153	0.488	0 / 2000
		154	0.415	0 / 2000
		155	0.336	1 / 2000
	F	156	0.408	1 / 2000
		157	0.328	1 / 2000
		158	0.374	1 / 2000
		159	0.413	1 / 2000
		160	0.438	1 / 2000
DMBPC
250	M	161	0.473	1 / 2000
		162	0.312	1 / 2000
		163	0.272	0 / 2000
		164	0.288	1 / 2000
		165	0.411	0 / 2000
	F	166	0.342	1 / 2000
		167	0.320	1 / 2000
		168	0.328	0 / 2000
		169	0.333	0 / 2000
		170	0.314	0 / 2000

 

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.

Executive summary:

The potential of the test material to cause in vivo genotoxicity was investigated in a micronucleus assay conducted in accordance with the standardised guideline OECD 474 under GLP conditions.

Male and female ICR mice were exposed to the test material via the intraperitoneal route in corn oil. Following a pilot study and toxicity assay, for the definitive micronucleus assay mice were assigned to seven groups, each containing 5 males and 5 females. Animals in five of these groups were treated either with the controls (negative or positive) or with DMBPC at a dose of 62.5, 125 or 250 mg/kg and were euthanised 24 hours after treatment. Animals in the other two groups were treated either with the negative control or DMBPC at a dose of 250 mg/kg and were euthanised 48 hours after treatment. Mice were observed after dose administration for clinical signs of toxicity.

Immediately following sacrifice, the femurs were distally exposed, cut just above the knee, and the bone marrow collected and processed. Two slides for examination were prepared from each mouse. Bone marrow cells [polychromatic erythrocytes (PCEs) and normochromatic erythrocytes (NCEs)], were analysed for the presence of micronuclei; 2000 polychromatic erythrocytes per animal were scored for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated for each animal. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes (PCEs/ECs ratio).

In the definitive micronucleus test, no mortality occurred at any dose level during the course of the micronucleus study. Clinical signs following dose administration included: lethargy and piloerection in all male mice and all female mice at 250 mg/kg. All other animals treated with the test or control articles appeared normal following dose administration.

Slight to moderate reductions of 12 to 33 % in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. The number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in test article-treated groups was not statistically increased relative to the respective vehicle controls in either male or female mice, regardless of dose level or bone marrow collection time. CP induced a statistically significant increase in micronucleated polychromatic erythrocytes in both male and female mice. All criteria for a valid test were met.

Under the conditions of this study, a single intraperitoneal administration of DMBPC at doses up to 250 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, DMBPC was concluded to be negative in the micronucleus test using male and female ICR mice.