α,α'-propylenedinitrilodi-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

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

α,α'-propylenedinitrilodi-o-cresol

EC Number:

202-374-2

EC Name:

α,α'-propylenedinitrilodi-o-cresol

Cas Number:

94-91-7

Molecular formula:

C17H18N2O2

IUPAC Name:

α,α'-propylenedinitrilodi-o-cresol

Details on test material:

- Test Item: Alpha,alpha'-Propylenedinitrilodi-o-cresol
- BASF Test Item No.: 11/0600-1
- Batch Number: 11000129U0
- Purity: >99 corr. area %
- Expiration Date: February 04, 2013
- Physical state, appearance: Liquid, highly viscous, yellowish
- Storage conditions: Room temperature

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean value 35.0 g (SD +- 1.8 g)
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: for a minimum of five days after their arrival

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +- 2 °C
- Humidity (%): 45 - 65 % (> 95% for few hours)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: [polyethylene glycol]
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
The administered volume was 10 mL/kg b.w. including test substance.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in PEG 400. The vehicle was chosen due to its relative non-toxicity for the animals. All animals received a single standard volume orally.

Duration of treatment / exposure:

single oral application

Frequency of treatment:

once

Post exposure period:

24 or 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 males for the test substance, 5 males for controls

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration potent inducer of micronuclei
- Doses / concentrations: 40 mg/kg bw.

Examinations

Tissues and cell types examined:

Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.
The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animal’s body weight. The animals received the test item, the vehicle, or the positive control substance once orally. Seven males were treated per dose group and sampling time. However, one animal of the highest dose group (animal no. 21) died 24h after administration. Five males each were treated for each vehicle and the positive control group. The animals of all dose groups, except the positive control were examined for acute clinical signs at intervals of around 1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.

DETAILS OF SLIDE PREPARATION and METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
All animals per sex and test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met:
- at least 5 animals per test group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the vehicle control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the vehicle control.

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statistical methods (nonparametric Mann-Whitney test (8)) are used as an aid in evaluating the results, if necessary. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test are used as an aid in evaluating the results, if necessary.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality in one top dose animal

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
Two animals of each sex treated in the pre-experiments received the test item Alpha,alpha`-Propylenedinitrilodi-o-cresol dissolved in PEG 400 once orally. The volume administered was 10 mL/kg b.w..
In the first pre-experiment a dose of 1500 mg/kg b.w. and in the second pre-experiment a dose of 2000 mg/kg b.w. was tested in males and females. In both pre-experiments no clinical signs of toxicity were observed.
On the basis of these data 2000 mg/kg b.w. were estimated to be suitable as highest dose.
No sex specific differences were observed with regard to clinical signs. In accordance with the test guidelines the main study was performed using males only.


RESULTS OF DEFINITIVE STUDY
In the main experiment for each test item dose groups 7 males received once orally administrations of Alpha,alpha`-Propylenedinitrilodi-o-cresol dissolved in PEG 400. The volume administered was 10 mL/kg b.w.. Clinical signs of toxicity (Reduction of spontaneous activity, Abdominal position, Eyelid closure and Ruffled fur) were only observed in one high dose animal that died within 24 hours. All other dosed animals were free of clinical signs. The animals treated with the negative control (PEG 400) did not express any clinical signs.

After treatment with the test item at 48h preparation interval the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Alpha,alpha`-Propylenedinitrilodi-o-cresol did not induce cytotoxic effects in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Alpha,alpha`-Propylenedinitrilodi-o-cresol were below or near to the value of the vehicle control group. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages and all values in dose groups were very well within the laboratory’s historical vehicle control data.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

The test item Alpha,alpha`-Propylenedinitrilodi-o-cresol was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was dissolved in PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w.. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. For the high dose group (24h) only six males were evaluated for the occurrence of micronuclei, as one mouse died intercurrently. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000, and 2000 mg/kg b.w.
48 h preparation interval: 2000 mg/kg b.w.

The highest dose was estimated by a pre-experiment to be suitable.

After treatment with the test item at 48h preparation interval the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that Alpha,alpha`-Propylenedinitrilodi-o-cresol did not induce cytotoxic effects in the bone marrow. However, one animal (no. 21) of the highest dose group showed clinical signs and died approximately 24h after administration indicating systemic exposure.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with Alpha,alpha`-Propylenedinitrilodi-o-cresol were below or near to the value of the vehicle control group. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages and all values in dose groups were very well within the laboratory’s historical vehicle control data.

40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item Alpha,alpha`-Propylenedinitrilodi-o-cresol did not induce micronucleias determined by the micronucleus test in the bone marrow cells of the mouse.

Applicant's summary and conclusion

Conclusions:

During the study described and under the experimental conditions reported, the test item Alpha,alpha`-Propylenedinitrilodi-o-cresol did not induce micronucleias determined by the micronucleus test in the bone marrow cells of the mouse.