p-(1,1-dimethylpropyl)phenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information

Two Ames tests, one MLA test and an in vivo bone marrow micronucleus assay.

Link to relevant study records

Reference

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:

1990-04-11 to 1990-05-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: study well conducted according to EPA guideline

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals were obtained from Charles River Breeding Laboratories (UK), Margate, Kent, England. All animals were acclimatised for at least four days
prior to treatment.
The animals were held in a room kept at positive pressure with respect to the outside. The room had its own supply of filtered, fresh air which is
passed to atmosphere and not recirculated. Temperature and humidity controls were designed to maintain conditions of 21 +/- 2 degreesC and 55%=/-15% relative humidity. There are 15 air changes per hour and a 12-hour light:12-hour dark cycle was in operation.

Route of administration:

oral: unspecified

Vehicle:

Maize oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The dosing solution was 10 ml/kg.

Duration of treatment / exposure:

Five male and five female mice per group were killed 24 hours after treatment; further lots of five males and five females from
groups 1 and 4 (males) or 5 (females) were killed 48 and 72 hours after treatment.

Frequency of treatment:

Dose administered to each animal once only.

Remarks:

Doses / Concentrations:
0, 62.5, 250, 1000, 4000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

M= male, F = female.
Group 1 Vehicle control (15 M, 15F)
Group 2 Dose 62.5 mg/kg = 5M
Group 3 Dose 250 mg/kg = 5M, 5F.
Group 4 Dose 1000 mg/kg = 15M, 5F.
Group 5 Dose 4000 mg/kg = 15F
Group 6 Positive control = 5M, 5F.

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil - 30 mg/kg in aqueous 10% ethanol

Tissues and cell types examined:

Bone marrow from the femur

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses and sex selection for the main study were selected from a preliminary toxicity test. Two males treated with
Nipacide PTAP at 2000 and 5000 mg/kg showed adverse reactions to treatment including hunched posture, piloerection, lethargy, prone posture,
laboured breathing, irregular breathing and rales. The signs were observed from the day after dosing and the mice were sacrificed in extremis when the signs became severe on the second or third day of the treatment period. Two female mice treated at 5000 mg/kg showed adverse reactions to
treatment including lethargy, hunched posture and piloerection. The signs were observed on the second and third days of the treatment period, but were absent on the fourth day just before the scheduled sacrifice of the mice. No adverse reactions to treatment were observed in mice treated with
Nipacide PTAP at the lower dosages.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Femurs were removed from sacrificed animals 24, 48 and 72 hours post dosing.Femurs were rapidly dissected out and cleaned of adherent tissue. The epiphyses were cut off to obtain access to the marrow canal.
Marrow cells were flushed out with 2.5 ml foetal calf serum using a syringe and needle. The recovered cells were centrifuged at 1000 rpm for five
minutes. The bulk of the supernatant fluid was discarded and the cell pellet resuspended in the remaining fluid.

DETAILS OF SLIDE PREPARATION: Single drops of the cell suspension were transferred to clean, dry slides, two or three smears (for the preliminary
toxicity test or main micronucleus test respectively) prepared, and the slides left to air-dry. Following fixation in methanol for ten minutes, they werestained manually, using the Schmid (May-Grunwald and Giemsa) staining technique. When air-dried, permanent mounts were made using DPX mountant, after clearing for five minutes in xylene.


METHOD OF ANALYSIS: The slides were examined under the light microscope, and regions judged to be of adequate technical quality to permit
scoring were selected under low magnification. At high magnification (x 1000, oil immersion) a total of at least 2000 erythrocytes per animal were
examined. Each erythrocyte scored was classed and polychromatic or mature: polychromatic cells stain blue/pink and the older cells stain red/pink.At least 1000 cells o f each type were scored from each animal where possible, but where there was an appreciable deviation from unity in the ratio
of polychromatic to mature erythrocytes, scoring continued until a minimum of 2000 of the predominant cell type were counted. Each erythrocyte
scored was examined for the presence or absence of micronuclei.

OTHER:

Evaluation criteria:

The number of micronucleated cells per 1000 erythrocytes was calculated for each animal. The ratio of polychromatic to mature cells was also
determined; a decrease in this may indicate inhibition of cell division following treatment, and the incidence of micronuclei in the mature cell
population 24 hours after treatment reflects the pretreatment situation, since most of these cells were produced before treatment. The frequency of micronuclei in polychromatic cells provides an index of induced genetic damage.

Statistics:

The frequencies of micronucleated cells per 1000 polychromatic erythrocytes scored were subjected to statistical analysis by the
Mann-Whitney procedure (Mann and Whitney, 1942). A computer-based version of this test was employed and significance was determined
by reference to tabulated values of R1. Data from males and females within each group were compared using a two-tailed test. Data from each
treatment group were compared with corresponding vehicle control group values, using a one-tailed test, for male and female animals separately.
[Mann, H.B. and Whitney, D.R. (1942) On a test fo whether one or two random variables is stochastically larger than the other. Ann Math Statist, 18, 50-60.]

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Hunched posture, piloerection and rales; 2 males; 1000 mg/kg dose.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Sex:

female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Hunched posture, piloerection and rales; 4 females; 4000 mg/kg dose.

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Males:
Among male mice killed 24 hours after treatment, the mean incidence of micronucleated polychromatic erythrocytes (per 1000 polychromatic cells
scored) was 1.4 for the vehicle control group, with a range of 1.0-2.0. Corresponding values for male animals given Nipacide PTAP at 62.5, 250 or
1000 mg/kg were closely similar: 1.1, 0.5 or 1.2 with ranges of 0.0-2.8, 0.0-1.7 and 1.0-1.9 respectively.

Among male mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in the vehicle control groups
were 1.3 (range 0.0-3.8) and 0.2 (range 0.0-1.0) respectively. Corresponding values for male mice given Nipacide PTAP at 1000 mg/kg were 0.8
(range 0.0-1.0) and 1.2 (range 0.0-2.0) respectively.

Females:
For female animals at 24 hours, the mean incidence of micronucleated polychromatic erythrocytes for the vehicle control group was 0.6, with a range of 0.0-1.0. Corresponding values for female animals given Nipacide PTAP at 250, 1000 or 4000 mg/kg were closely similar: .3, 0.8 or 0.6 with ranges of 0.9-2.0, 0.0-3.0 and 0.0-2.0 respectively.

Among female mice killed after 48 or 72 hours, the mean incidences of micronucleated polychromatic erythrocytes in the vehicle control groups
were 0.2 (range 0.0-0.9) and 0.6 (range 0.0-1.9) respectively. Corresponding values for females given Nipacide PTAP at 4000 mg/kg were 0.7 (range
0.0-1.9) and 0.4 (range 0.0-1.0) respectively.

Chlorambucil treatment produced a range of micronucleated cells per 1000 polychromatic erythrocytes from 59.6-133.1

with a mean of 95.7. Statistical analysis showed that animals treated with chlorambucil had significantly more micronucleated polychromatic cells than vehicle controls (p < 0.01). This increase in chromosomal damage after exposure to a known mutagen demonstrates the sensitivity of the test system.

Conclusions:

Interpretation of results (migrated information): negative
Nipacide PTAP, under the conditions of the test, did not induce chromosomal or other damage leading to micronucleus formation in polychromatic
erythrocytes in the bone marrow of mice treated once by the oral route and killed 24, 48 or 72 hours later.

Executive summary:

In a bone marrow micronucleus assay, P-tert amylphenol was administered orally to CD1 strain mice (n=5 males and n=5 females per dose level) at dose levels of 0, 62.5, 250, 1000, 4000 mg/kg (females) and 0, 62.5, 250, 1000 mg/kg (males) in maize oil. Under the conditions of this test, P-tert amylphenol did not induce chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes in the bone marrow of mice treated once by the oral route and killed 24, 48 or 72 hours later. P-tert amylphenol is not mutagenic under the conditions of this study. As a general observation in this study, signs of hunched posture, piloerection and rales were seen in 2 males at the 1000 mg/kg dose and in 4 females at the 4000 mg/kg dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:  

There were no adverse findings in any of the battery of in vitro tests. In the definitive study, ie an in vivo bone marrow micronucleus assay, p-tert amylphenol was administered orally to CD1 strain mice (n=5 males and n=5 females per dose level) at dose levels of 0, 62.5, 250, 1000, 4000 mg/kg (females) and 0, 62.5, 250, 1000 mg/kg (males) in maize oil. Under the conditions of this test, p-tert amylphenol did not induce chromosomal or other damage leading to micronucleus formation in polychromatic erythrocytes in the bone marrow of mice treated once by the oral route and killed 24, 48 or 72 hours later.

Justification for selection of genetic toxicity endpoint  OECD guideline in vivo study.

Justification for classification or non-classification

Not mutagenic.

No positive or ambigous results have been observed in any test of an adequate testing battery.