α,α'-propylenedinitrilodi-o-cresol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (BASF SE, 2012): negative
HPRT-Assay (Harlan, 2012): negative

Ames test

The test substance alpha,alpha’-Propylenedinitrilodi-o-cresol was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay according to OECD 471 guideline and GLP.

STRAINS: TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA

DOSE RANGE: 1 μg - 5 000 μg/plate (SPT, Salmonella strains)

33 μg - 5 000 μg/plate (SPT, E. coli)

1 μg - 333 μg/plate (PIT, Salmonella strains)

10 μg - 2 500 μg/plate (PIT, E. coli)

TEST CONDITIONS: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (liver S9 mix from induced rats).

SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 33 μg/plate onward. MUTAGENICITY: A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

CONCLUSION: Thus, under the experimental conditions of this study, the test substance alpha,alpha’- Propylenedinitrilodi-o-cresol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Additional data from an AmesII test is available (BASF, 1999) which also shows that the test substance is not mutagenic in several bacteria strains.

HPRT-Assay:

A GLP-compliant gene mutation assay, tested according to OECD guideline 476, was performed to investigate the potential of Alpha,alpha’-Propylenedinitrilodi-o-cresol to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (Harlan 2012).

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.The concentration range of the main experiments was limited by cytotoxic effects of the test item.The test item was dissolved in DMSO.Relevant cytotoxic effects occurred in the first experiment at 88.0μg/mL without metabolic activation and at 264μg/mL with metabolic activation. In the second experiment cytotoxic effects occurred at 176μg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation. Precipitation of the test item was noted at 88.0μg/mL and above in experiment I with metabolic activation. However, the precipitate was probably denatured protein rather than test item per se as there was no precipitation in the second experiment at comparable or even higher concentrations.No substantial and reproducible dose dependent increase of the mutation frequency was observed up to the maximum concentration with and without metabolic activation.Therefore, Alpha,alpha’-Propylenedinitrilodi-o-cresol is considered to be non-mutagenic in this HPRT assay.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS/TRP

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

1 μg - 5 000 μg/plate (SPT, Salmonella strains)
33 μg - 5 000 μg/plate (SPT, E. coli)
1 μg - 333 μg/plate (PIT, Salmonella strains)
10 μg - 2 500 μg/plate (PIT, E. coli)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was used as vehicle, which
had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: With S9 mix: 2-aminoanthracene (2-AA); Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Incubation period: 20 min in medium (PIT) and incubation at 37°C for 48 – 72 hours (PIT and SPT)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.
A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

not applicable

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

test conditions from about 33 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

test conditions from about 33 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants, reduction in the titer) was observed in the standard plate test depending on the strain and test conditions from about 100 μg/plate onward. In the standard plate test with
E.coli WP2uvrA a bacteriotoxic effect (reduced trp- background growth, decrease in the number of trp+ revertants, reduction in the titer) was observed from about 1 000 μg/mL onward.
In the preincubation assay bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants, reduction in the titer) was observed depending on the strain and test conditions from about 33 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

According to the results of the present study, the test substance did not lead to a relevant increase in the number of revertant colonies either without S9 mix or after adding a metabolizing system in three experiments carried out independently of each other (standard plate test and preincubation assay.

Besides, the results of the negative as well as the positive controls performed in parallel corroborated the validity of this study, since the values fulfilled the acceptance criteria of this study.

In this study with and without S9 mix, the number of revertant colonies in the negative controls was within or nearby the range of the historical negative control data for each tester strain.

In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data or above.

Conclusions:

Under the experimental conditions of this study, the test substance alpha,alpha’- Propylenedinitrilodi-o-cresol is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase)

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

Before freezing, the level of spontaneous mutants was depressed by treatment with HAT-medium. Each batch is screened for mycoplasm contamination and checked for karyotype stability and spontaneous mutant frequency.

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

See: Any other information on materials and methods.

Vehicle / solvent:

DMSO
The final concentration of DMSO in the culture medium was 0.5% v/v.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 7,12-dimethylbenz(a)anthracene

Remarks:

With metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: In the first experiment the treatment period was 4 hours with and without metabolic activation. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation.

- Expression/fixation time: Three or four days after treatment 1.5x10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5x10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

NUMBER OF REPLICANTS: The study was performed in two independent experiments, using identical experimental procedures.

DETERMINATION OF CYTOTOXICITY
- Method: Toxicity of the test item is indicated by a reduction of the cloning efficiency (CE).

Evaluation criteria:

The gene mutation assay is considered acceptable if it meets the following criteria:
The numbers of mutant colonies per 10^6 cells found in the solvent controls falls within the laboratory historical control data.
The positive control substances should produce a significant increase in mutant colony frequencies.
The cloning efficiency II (absolute value) of the solvent controls should exceed 50 %.
The data of this study comply with the above mentioned criteria

Evaluation of Results
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.

A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item visible to the naked eye was noted at 88.0 μg/mL and above in experiment I with metabolic activation. However, the precipitate was probably denatured protein rather than test item per se as there was no precipitation in the second experiment at comparable or even higher concentrations.

RESULTS GENOTOXICITY:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiments up to the maximum concentration. The mutant frequency generally did not exceed the historical range of solvent controls. A single increase of the induction factor exceeding the threshold of three times the mutation frequency of the corresponding solvent control was observed in the first culture of the second experiment without metabolic activation at 2.8 μg/mL (24 hours treatment). However, the increase was based on a rather low mutation frequency of the solvent control of just 4.8 colonies per 10^6 cells. Furthermore, the effect was not reproduced in the parallel culture under identical experimental conditions. Therefore, the increase of the induction factor was judged as biologically irrelevant fluctuation.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the second culture of the second experiment without metabolic activation. This trend however, was judged as irrelevant since it was not reproduced in the parallel culture under identical experimental conditions and the threshold described above was not exceeded.

RANGE-FINDING/SCREENING STUDIES:
The range finding pre-experiment was performed using a concentration range of 22.1 to 2820 μg/mL (≈10 mM) to evaluate toxicity in the presence (4 hours treatment) and absence (4 hours and 24 hours treatment) of metabolic activation.
Relevant cytotoxic effects indicated by a relative suspension growth below 50 were noted at 176.3 μg/mL and above in the presence of metabolic activation following 4 hours treatment. Strong toxic effects indicated by a completely inhibited cell growth occurred in the absence of metabolic activation at 176.3 μg/mL and above following 4 hours treatment and at 44.1 μg/mL and above following 24 hours treatment.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) prior to removal to the test item. Precipitation occurred at 1410 μg/mL and above with and without metabolic activation following 4 and 24 hours treatment.
There was no relevant shift of the osmolarity of the medium even at the maximum concentration of the test item measured in the pre-experiment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative cloning efficiency I or cell density below 50% in both parallel cultures occurred in the first experiment at 88.0 μg/mL without metabolic activation and at 264 μg/mL with metabolic activation. In the second experiment cytotoxic effects as described above occurred at 176 μg/mL and above with metabolic activation. The recommended cytotoxic range of approximately 10-20% relative cloning efficiency I or relative cell density was covered with and without metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

The substance is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus Assay (Harlan, 2013): negative

A study was performed to investigate the potential of Alpha,alpha`-Propylenedinitrilodi-o-cresol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse according to OECD 474 guideline and GLP (Harlan, 2013).

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, based on results of a pre-experiment:

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

After treatment with the test item 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 exert a cytotoxic effect in the bone marrow. However, one animal (no. 21) of the highest dose group showed clinical signs and died approximately 24h after administration.

In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency.

In conclusion, it can be stated that under the experimental conditions reported, the test itemAlpha,alpha`-Propylenedinitrilodi-o-cresol did not induce micronucleias determined by the micronucleus test with bone marrow cells of the mouse. Therefore, Alpha,alpha`-Propylenedinitrilodi-o-cresol is considered to be non-mutagenic in this micronucleus assay.

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

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

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

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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.

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.

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.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In Vitro Mammalian Chromosome Aberration Test

The test item Alpha,alpha’-Propylenedinitrilodi-o-cresol was assessed for its potential to induce structural chromosome aberrations in mammalian cells (V79 cells of the Chinese hamster) in vitro in one experiment according to OECD 473 guideline and GLP. In each experimental group (solvent control, test item (11 -176 µg/mL) and positive control) two parallel cultures were set up. 100 metaphases per culture were evaluated for structural chromosome aberrations.

In the absence of S9 mix concentrations showing clear cytotoxicity were not scorable for cytogenetic damage. In the presence of S9 mix clear cytotoxicity was observed at the highest evaluated concentration. Clastogenicity was observed in the absence of S9 mix after treatment with 22.0, 44.1 and 88.1 µg/mL (13.5, 12.5, 14.0 % aberrant cells, excluding gaps) and in the presence of S9 mix after treatment with 22.0, 44.1, 88.1 and 176.3 µg/mL (10.5, 7.0, 10.0 and 20.5 % aberrant cells, excluding gaps) clearly exceeding the range of the historical control data of 0.0 - 4.0 % aberrant cells, excluding gaps. No relevant increase in polyploid metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. No relevant increase in endomitotic metaphases was found after treatment with the test item as compared to the frequencies of the control cultures. Appropriate mutagens (EMS and CPA) were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro. Therefore, Alpha,alpha’-Propylenedinitrilodi-o-cresol is considered to be clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest evaluable concentration.

Justification for classification or non-classification

Negative results were obtained with an Ames-Test and with a HPRT-Test. The positive in vitro results in a chromosome aberration test could not be confirmed in an in vivo Micronucleus Assay. Threfore no classification for mutagenicity is required according to EU classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.