2,2'-methylenebis(6-nonyl-p-cresol)

Genetic toxicity: in vivo

Administrative data

Endpoint:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Study period:

1 December 1987 - 21 March 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)

Deviations:

no

Remarks:

The study pre-dates the guideline but follows and equivalent study protocol.

GLP compliance:

yes

Type of assay:

chromosome aberration assay

Test material

Test material information

Constituent 1

Reference substance name:

6,6'-di-tert-butyl-4,4'-thiodi-m-cresol

EC Number:

202-525-2

EC Name:

6,6'-di-tert-butyl-4,4'-thiodi-m-cresol

Cas Number:

96-69-5

IUPAC Name:

4,4'-sulfanediylbis(2-tert-butyl-5-methylphenol)

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Container: clear, colourless glass bottle.
- Appearance: white powder.
- Storage conditions: at room temperature in the container received from the sponsor.

Test animals

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 61 days.
- Weight at study initiation: males 139 - 167 g; females 101 - 117 g.
- Assigned to test groups randomly: Yes; randomly assigned to experimental groups by initial ranking by body weight and the use of random numbers
- Fasting period before study: Yes, approximately 18 hours.
- Housing: Rats were housed individually in stainless steel wire mesh cages in accordance with the "Guide for the Care and Use of Laboratory Animals" of the Institute of Laboratory Resources. Waste material was removed two times a week.
- Diet: ad libitum.
- Water: fresh tap water was available ad libitum using an automatic watering system.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- All required dilutions of the test article were prepared in corn oil prior to dosing.
- All rats were administered a single oral dose at a constant dose volume of 10 mL/kg.

Details on exposure:

- Weighed amounts of the test material were ground with a mortar and pestle and diluted in corn oil to the designated dose level. Homogeneous suspensions were obtained and maintained on a magnetic stir plate during the dosing phase.

Duration of treatment / exposure:

After dosing, animals were sacrificed at 6, 18 or 30 hours.

Frequency of treatment:

A single oral dose.

Post exposure period:

Up to 30 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
700 and 1400 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

5 animals per sex per dose per sampling time.

Control animals:

yes, concurrent vehicle

Positive control(s):

A positive control group was treated with cyclophosphamide at 20 mg/kg bw. Formulated in distilled water.

Examinations

Tissues and cell types examined:

Haemopoietic cells of the bone marrow cells present in the femur.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
Dose Selection: Based on the results of a preliminary study (see “Additional information on results”) and in discussion with the sponsor, a dose of 1400 mg/kg was selected as an estimate of the maximum tolerated dose for evaluation in the Metaphase Analysis Assay. One additional dose of 700 mg/kg (half of the maximum tolerated dose) was also evaluated.

TREATMENT AND SAMPLING TIMES
Two to three hours prior to sacrifice, each animal was given a single intraperitoneal dose of colchicine at 4 mg/kg of body weight to arrest dividing cells in metaphase. Colchicine was dosed at 10 mL/kg of body weight and was prepared in distilled water.
Both dose levels were sampled at 6, 18 and 30 hours after treatment.

Bone Marrow Cell Harvest
Approximately two to three hours after injection of colchicine, animals were sacrificed by CO2 inhalation. Immediately after sacrifice both femurs were removed and the surrounding muscle tissue cleaned away. The distal end was snipped off one bone and the proximal end off the other bone. Bone marrow cells were then flushed into appropriately labelled 15 mL centrifuge tubes containing 10 mL of pre-warmed (37 °C) phosphate buffered saline (PBS) pH 7.0. A 1 cc syringe with a 22 gauge needle was used to flush and disperse the bone marrow cells in the PBS. Cells were sedimented at 1000 rpm for 5 minutes in an IEC Centra-TR refrigerated centrifuge, the supernatant decanted and 10 mL of warmed (37 °C) hypotonic KCl (0.075 M) was dispensed to each tube. Cells were resuspended and kept in the hypotonic solution for approximately 15 minutes to swell the cells. Cells were sedimented again, the supernatant decanted and 10 mL of freshly prepared fixative (3 parts methanol:1 part glacial acetic acid) was dispensed to each tube and cells were resuspended. Cells were washed an additional two times in 5 mL washes of fixative. The total fixation time was a minimum of 30 minutes. Cells were stored refrigerated in 5 mL of fixative for slide preparation at a later time.

DETAILS OF SLIDE PREPARATION
Cells were resuspended in a small amount of freshly prepared fixative. Slides were prepared by dropping the cell suspension on precleaned methanol-wet glass slides followed by flaming. Slides were stained in 3 % Giemsa in distilled water for 10 minutes, rinsed in distilled water and air dried. Slides were cleared in xylene and coverslips mounted with Permount.

METHOD OF ANALYSIS
A total of 500 well spread, intact metaphase cells (if possible) were scored for the presence of chromosome aberration per experimental treatment point (50 per animal) by two investigators (25 each per animal). Cells were located by systematic searching of the slide under low power (20 - 40 X) magnification. Cells judged acceptable for analysis based on cell morphology and total centromere number (± 2 of the normal diploid no. of 42) were then further analysed with a 100 X oil immersion objective where abnormalities were detected and classified. Vernier coordinates were recorded for the first and last metaphase scored, as well as for any abnormal metaphases (including gaps) observed. The centromere number was recorded for all cells analysed. All slides were scored for chromosome damage using a Nikon Optiphot microscope.

Evaluation criteria:

Cytogenetic Analysis: Cytogenetic abnormalities were classified on a standard scoring sheet according to chromosome or chromatid aberrations and further according to type of aberration. Aberrations were classified according to the nomenclature of Buckton and Evans, 1973 and Savage, 1975.

Statistics:

Mean number of aberrations per cell per rat (50 cells per rat) were analysed for statistically significant increases in chromosome aberration by a one-way analysis of variance (ANOVA). Each sampling time was analysed separately as compared to its concurrent vehicle control group. The positive control group was not included in the ANOVA. Data from this group were analysed separately by a one-tailed t test comparing it with the 18 hour vehicle control. The mean and standard deviation of aberrations/cell were also determined for each group of rats (500 cells; 50 cells per rat). The number of aberrant metaphases was analysed by Chi-square analysis for statistically significant increases. Statistical significance was determined at the p≤0.05 probability level.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

see below

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

Procedure
A preliminary study was done to determine if rats would survive oral treatment with the test material at 2075 mg/kg (half of the oral LD50 in rats) and at 1500 mg/kg. Healthy young adult male and female Fisher 344 rats (55 days of age for high dose group; 62 days of age for low dose group) were used for this assay. Two male and two female rats fasted for approximately 18 hours were administered the test material at 1500 or 2075 mg/kg in a single dose by oral gavage. Animals were observed for pharmacotoxic signs immediately and one hour after dosing. No pharmacotoxic signs were observed at these times. Animals were again weighed and observed on days 1, 3 and 7 at which time the remaining animals were sacrificed.

Day 1
-All animals administered the test material at 1500 mg/kg exhibited decreased body tone and activity, abnormal gait, abnormal stance, diarrhoea, piloerection and arched back. The males at this time lost an average of 3 g from the initial fasted weight while the females gained an average of 2.5 g.
- All animals administered the test material at 2075 mg/kg exhibited abnormal gait, abnormal stance, diarrhoea and decreased body tone. Both females exhibited piloerection with one female also exhibiting a decrease in activity. Males at this time had lost an average of 3 g from the initial fasted weight while females had a 6.5 g average loss.
Day 3
-All animals administered the test material at 1500 mg/kg exhibited decreased body tone, decreased activity, abnormal gait and abnormal stance, with both males and one female also exhibiting diarrhoea, piloerection and arched back. Males lost an additional average of 6 g and females lost an average of 2.5 g from the day 1 weights.
- All animals administered the test material at 2075 mg/kg had an abnormal gait and stance, diarrhoea, piloerection, decreased body tone and decreased activity. One male and both females exhibited rear limb extension and arching of the lower back. Males lost an average of 13.5 g and females lost an average of 8 g from the day 1 weights.
Day 6
-From the group administered the test material at 2075 mg/kg one male and two females were dead. Gross necropsies of these animals revealed stomach and caecum which were dark red throughout with haemorrhagic areas. The lungs, thymus and adrenals were dark red and the intestines were discoloured, distended and fluid filled (dark red to brown in colour). The dead male lost 9 g from day 3 giving a total net weight loss of 31 grams from the initial fasted weights. The dead females lost an average of 12.5 g from day 3 giving a net average weight loss of 27 g.
Day 7
-All animals administered the test material at 1500 mg/kg exhibited decreased body tone, abnormal gait, decreased activity and arched back. Both males exhibited piloerection and one male also had an abnormal stance and exhibited poor grooming. One male and one female exhibited enophthalmus. Males gained an average of 30 g from day 3 giving a total average net gain of 21 g from the initial fasted weight. Females gained an average of 12 g from day 3 with a total average net gain of 12 g from the initial fasted weights.
-From the group administered the test material at 2075 mg/kg, the remaining male exhibited decreased activity, decreased body tone, abnormal gait, abnormal stance and arched back. This animal gained 18 g from day 3 giving a net gain of 7 g from the initial fasted weight.



RESULTS OF DEFINITIVE STUDY

PHARMACOTOXIC EFFECTS OF TREATMENT
Rats were observed immediately after dosing and again just prior to colchicine administration.
-Immediately after dosing no pharmacotoxic signs were observed in any group of rats.
-Prior to colchicine administration, the following pharmacotoxic signs were observed:

6 Hour Group (observed approximately 4 hours after dosing)
700 mg/kg dose level - All rats exhibited decreased body tone, diarrhoea, abnormal gait, piloerection and brown discoloration around the oral-nasal region and forepaws. All females also exhibited decreased activity. Males in this group had an average body weight gain of 0.6 g from the initial fasted body weights. Females had an average gain of 1.8 g.
1400 mg/kg dose level - All rats exhibited decreased body tone, decreased activity, piloerection, abnormal gait, abnormal stance, arched back, diarrhoea, and brownish discoloration around the oral-nasal region and forepaws. Males at this time had an average weight loss of 4.4 g as compared to the original fasted weight while females lost 1.2 g on average.

18 Hour Group (observed approximately 16 hours after dosing)
700 mg/kg dose level - All rats exhibited decreased body tone, diarrhoea, abnormal gait, arched back and brown discoloration around the nasal-oral area and forepaws. Most of the animals also exhibited decreased activity and three females exhibited an abnormal stance. Males in this group lost an average of 3 g from the original fasted weight and the females lost an average of 2.2 g.
1400 mg/kg dose level - All rats exhibited diarrhoea, decreased body tone, abnormal gait, abnormal stance and brownish discoloration on forepaws and around the oral-nasal region. One female also had nasal rales, two females exhibited piloerection and two females had tremors. Males had an average weight loss of 7.8 g while females lost an average of 2.4 g as compared to the original fasted weights.

30 Hour Group (observed approximately 28 hours after dosing)
700 mg/kg dose level -All rats exhibited decreased body tone, diarrhoea, abnormal gait and piloerection. Most of the animals exhibited brownish discoloration around the oral-nasal region and on the forepaws and three males had an abnormal stance. Males in this group had an average weight loss of 0.2 g as compared to the original fasted weight while the females’ average body weight gain was 4.2 g.
1400 mg/kg dose level - All rats exhibited diarrhoea, abnormal gait, abnormal stance, decreased body tone, decreased activity, piloerection, and brownish discoloration on forepaws in the nasal-oral region. Most of the animals also exhibited arched back, one male exhibited chromodacryorrhea and three females exhibited poor grooming. Males in this group lost an average of 2.2 g and females had an average gain of 1.6 g as compared to the original fasted weights.

Vehicle Control
-Two males and one female in the 6 hour corn oil group exhibited diarrhoea prior to colchicine administration. Males in the 6 hour corn oil group had an average weight loss of 1.4 g while the females gained 0.6 g on average.
-In the 18 hour corn oil group, by sacrifice time, the males had an average weight gain of 6.4 g while the females gained an average of 10 g from the initial fasted weights.
-In the 30 hour corn oil group, males gained 9 g average and the females gained 6 g average.

Positive Control
Males (18 hours) had an average gain of 6.4 g and females gained an average of 7.6 g compared to the initial fasted weights.


SUMMARY OF METAPHASES SCORED
A total of 500 metaphases/group (50/rat) were analysed in all but three groups.
-In the 700 mg/kg 6 hour group, only 35 cells were scored for one animal and 19 cells for another due to an accidental spill of cell suspensions prior to slide preparation. Thus, a total of 454 metaphases were scored in this group.
In the 1400 mg/kg 30 hour group, only 28 cells were scorable for one animal giving a total of 478 metaphases in this group.
-In the positive control group, a total of 460 metaphases were scored.


PROPORTION OF CELLS WITH ABERRATIONS
The proportion of cells with one or more aberrations (1 aberration or greater) was analysed by Chi-square analysis by comparing the number of cells with aberrations versus the number of normal cells in each treatment group versus the vehicle control (Table 1). Each time of sacrifice was analysed separately and groups within each sacrifice time were compared individually to the vehicle control. Cells with gaps only were not considered aberrant for this analysis. A statistically significant increase (p≤0.01) was detected with the positive control group at the 18 hour sacrifice. No other significant differences were noted.


ANALYSIS OF ABERRATIONS PER CELL
The mean number of aberrations per cell per animal were analysed for statistically significant increases by a one-way ANOVA for each time interval and data are presented in Table 2. Data reflect the total number of aberrations seen in all cells scored per group. No statistically significant differences (p≤0.05) from the vehicle controls were detected by this analysis in animals treated with the test material. Table 3 reflects a further classification as to the types of damage seen during slide analysis and calculation of the total aberrations per groups of ten animals.

Animals treated with cyclophosphamide gave a statistically significant (p≤0.01) increase in the number of aberrations. In this group 139 metaphases were severely damaged. These metaphases could not accurately be scored due to multiple aberrations with tangles (134 metaphases) and/or partially shattered chromosomes (5 metaphases). This metaphase population comprised 23.2 % of all metaphases scored in the group. These cells were noted on the scoring sheets but were not included in the total of 460 metaphases analysed statistically.

Any other information on results incl. tables

Table 1 Proportion of Cells with One or More Aberrations

Dose (mg/kg)	Harvest Time (hours)	No. of Cells with One or More Aberrations	No. of Normal Cells	Mean Proportion of Aberrant Cells/Animal ± SD	% Aberrant Cells Per Group
Vehicle Control	6 18 30	2 2 2	498 498 498	0.004 ± 0.008 0.004 ± 0.008 0.004 ± 0.008	0.4 0.4 0.4
700	6 18 30	2 7 7	452 493 493	0.004 ± 0.013 0.014 ± 0.010 0.014 ± 0.021	0.4 1.4 1.4
1400	6 18 30	6 2 6	494 498 472	0.012 ± 0.017 0.004 ± 0.013 0.014 ± 0.016	1.2 0.4 1.3
Positive Control	18	353*	107	0.769 ± 0.095	76.7

*significant in Chi-square at p≤0.01

 

Table 2 Mean Aberrations/Cell/Group

Dose (mg/kg)	Harvest Time (hours)	No. Metaphases Analysed	Total Aberrations Per Group	Mean Aberrations/Cell ± SD†
Vehicle Control	6 18 30	500 500 500	2 2 2	0.004 ± 0.008 0.004 ± 0.008 0.004 ± 0.008
700	6 18 30	454 500 500	2 7 7	0.004 ± 0.013 0.014 ± 0.010 0.014 ± 0.021
1400	6 18 30	500 500 478	6 2 6	0.012 ± 0.017 0.004 ± 0.013 0.014 ± 0.016
Positive Control	18	460	1719	3.898 ± 1.603*

†Calculated from the mean aberrations/cell for each animal in the group.

*Statistically significant (p≤0.01)

 

Table 3 Analysis of Chromosome Aberrations

Dose (mg/kg)	Harvest Time (hours)	No. Metaphases Scored	Chromatid Aberrations						Chromosome Aberrations				Total Aberrations
			Gaps	Del	Iso	Exchanges			Del	Ring	Dicen	Misc
						Inter	Intra	Trir
Vehicle Control	6 18 30	500 500 500	2 2 2	2 2 2	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	2 2 2
700	6 18 30	454 500 500	1 4 2	1 7 4	0 0 0	1 0 0	0 0 0	0 0 0	0 0 2	0 0 0	0 0 1	0 0 0	2 7 7
1400	6 18 30	500 500 478	0 1 2	5 2 6	1 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	0 0 0	6 2 6
Positive Control	18	460	70	881	144	454	117	95	22	6	0	0	1719*

 *Statistically significant (p≤0.01)

Legend to Chromosome Aberrations

Gaps - They are achromatic lesions of different lengths along the arm of one chromatid (gap) or at the same locus of both chromatids (isogap), except for the secondary constriction on certain chromosomes. They are scored but not considered chromosomal aberrations.

Chromatid Aberrations

Del = Deletions (c) - A simple severance of the chromosome or chromatid that gives an acentric fragment(s) at telophase.

 

Chromatid Exchanges

Inter = Interchange (c/c) - Exchanges involving two or more lesions situated in the chromatids of different chromosomes (symmetrical and asymmetrical).

Intra = Intrachanges (c/c) - Exchanges resulting from lesions within the same chromosome. Intrachanges are either inter-arm intrachanges (c/c ter) where the lesions affect both arms of the same chromosome or intra-arm intrachanges (c/c) where the lesions affect the chromatids of the same arm of the same chromosome.

Trir = Triradial (i/c) - Isochromatid/Chromatid breaks which involves two chromosomes or Intra which involves both arms of the same chromosome.

 

Chromosome Aberrations

Del = Deletions - Include terminal (Term) and interstitial (Int) deletions. Terminal (C) deletion is the complete severance of the terminal region of a chromosome, which gives rise to a shortened chromosome and an acentric fragment, and is not associated with any obvious exchange process. The acentric fragment may lie free among the chromosomes of the metaphase spread. Interstitial deletion is the severance of one intercalary acentric chromosomal segment resulting in one chromosome appearing shorter and a pair of minutes or acentric rings.

Ring (C/C) - Ring shaped chromosomes resulting from an exchange between two breaks occurring on either side of the centromere of the same chromosome.

Dicen = Dicentric (C/C) - Asymmetrical interchange aberrations due to a breakage in each of two or more chromosomes followed by aberrant rejoining such that the proximal regions of the chromosomes become united, thus forming dicentric structure and an associated acentric fragment(s). 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test material was judged negative in its ability to induce structural chromosomal aberrations to the haemopoietic cells of the rat bone marrow under the experimental conditions of this assay.

Executive summary:

A study was conducted to evaluate the potential of the test material to induce structural chromosomal aberrations in the haemopoietic cells of rat bone marrow when administered by oral gavage. Though the study was carried out before the introduction of the standardised OECD guideline 475, the assay followed the same scientific principles.

The dose levels were based on the results of a preliminary toxicity test. The maximum tolerated dose was estimated to be 1400 mg/kg, and was used as the high dose in the definitive study. An additional dose level of 700 mg/kg was also evaluated as one-half of the maximum tolerated dose, and was used as the low dose level. The test material and the vehicle control (corn oil) were administered in single oral doses to three groups of two male and three female Fisher 344 rats per dose. Animals were sacrificed at 6, 18 and 30 hours after dosing. An extra group of rats was dosed with the positive control (Cyclophosphamide 20 mg/kg) and sacrificed 18 hours later. Approximately two hours prior to each sacrifice, animals were administered colchicine at 4 mg/kg of body weight to arrest cells in metaphase. At the appropriate time, animals were sacrificed and both femurs were removed from each animal and metaphase slides prepared. Slides were stained, coded and scored for chromosomal aberrations.

 

All rats dosed at 700 mg/kg and 1400 mg/kg exhibited mild to severe pharmacotoxic signs at all sacrifice time intervals evaluated. These observations suggest that the test material was tested near the maximum tolerated dose.

Statistical analysis of the data indicated that the test material did not produce statistically significant increases in the number of aberrations or in the number of aberrant metaphases at any of the three sacrifice times evaluated.

Therefore the test material was judged negative in its ability to induce structural chromosomal aberrations to the haemopoietic cells of the rat bone marrow under the experimental conditions of this assay.