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Toxicological information

Genetic toxicity: in vivo

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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:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
The objective of this study was to evaluate the potential of N-isopropyl-N'phenyl-p-phenylenediamine to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats and/or to induce DNA strand breaks in the liver, stomach and duodenum of the same animals using the alkaline comet assay.

GLP compliance:
yes
Type of assay:
other: Mammalian Erythrocyte Micronucleus Test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Purity: 97.6%
dark brown to black solid

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)
Details on species / strain selection:
The rodent bone marrow micronucleus test is recommended by various regulatory authorities as an appropriate test to determine the genotoxic potential of a compound in vivo (OECD 474, 2016). The rat was selected as there is a large volume of background data of this end-point for this species. The study was specifically requested by the European Chemicals Agency (ECHA) for REACH registration; the tissues and analysis selected comply with ECHA requirements.

45 male young adult out-bred Sprague Dawley rats Crl:CD(SD) were obtained from Charles River (UK) Ltd., Margate, UK.
Sex:
male
Details on test animals and environmental conditions:
Species, Strain, Supplier, and Specification
45 male young adult out-bred Sprague Dawley rats Crl:CD(SD) were obtained from Charles River (UK) Ltd., Margate, UK.
18 male animals were dosed during the Range-Finder Experiment. They were 7 to 11 weeks old and 227-420 g on the first day of dosing.
27 male animals were dosed during the Main Experiment. They were 7 to 9 weeks old and 260-322 g on the first day of dosing.
Environment
Animals were housed in wire topped, solid bottomed cages.
The animals were housed in rooms air-conditioned to provide 15-20 air changes/hour and set to maintain temperature and relative humidity in the range 20 to 24°C and 45 to 65%, respectively. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800) and 12 hours dark. The animals were routinely kept under these conditions except for short periods of time where experimental procedures dictated otherwise.

Diet, Water, Bedding and Environmental Enrichment
Throughout the study the animals had access ad libitum access to 5LF2 EU Rodent Diet 14% (manufactured by LabDiet St. Louis USA). Each batch of diet was analysed for specific constituents and contaminants.
Mains water was provided ad libitum via water bottles. The water supply was periodically analysed for specific contaminants.
Bedding was provided on a weekly basis to each cage by use of clean wood bedding (Aspen). The bedding was analysed for specific contaminants.
In order to enrich both the environment and the welfare of the animals, they were provided with wooden Aspen chew blocks and rodent retreats.
No contaminants were present in any of the above at levels that might interfere with achieving the objective of the study.

Allocation to Treatment Group
On arrival animals were randomly allocated to cages. Range-Finder were allocated to groups of up to three and Main Experiment animals were allocated to groups of six (three for the positive control(s)).
Checks were made to ensure the weight variation of Main Experiment animals prior to dosing was minimal and did not exceed ±20% of the mean weight.

Identification
The animals were individually identified by uniquely numbered tail mark (Range-Finder Experiment) or electronic transponder (Main Experiment). Cages were appropriately identified (using a colour-coded procedure) with study information including study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Test Article Formulation:
Preparation

All formulations for the main study were prepared as follows:
The test article was weighed and transferred to a mortar. A small volume of vehicle (arachis oil) was added and mixed with a pestle to form a smooth paste. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume. Formulations were then mixed using a Silverson until visibly homogenous.

Stability
Formulations of N-isopropyl-N'phenyl-p-phenylenediamine in arachis oil were prepared at 0.2, 1 and 100 mg/mL and analysed for stability as follows:
Duplicate samples were taken from the top, middle and bottom of each test article formulation and all samples were analysed for achieved concentration and a determination of homogeneity. The remaining bulk formulation was split into 2 aliquots; one aliquot was stored at room temperature and the other was stored at 2 8ºC. After 1 or 3 days storage, triplicate samples were taken from the middle of the room temperature aliquot, which were analysed for achieved concentration. No samples were taken from the aliquot held at 2-8ºC.

Homogeneity
To ensure homogeneity, dose formulations were stirred continuously (on a magnetic stirrer) immediately before and throughout dosing.

Formulations Analysis
Samples were taken from the 10 mg/mL formulation in arachis oil used in the Range-Finder Experiment and all test article formulations and vehicle control used in the Main Experiment.
Duplicate samples were taken from the top, middle and bottom of each test article formulation together with a single sample (taken from the middle) of the vehicle control and all samples were analysed for achieved concentration and a determination of homogeneity.

Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All formulations for the main study were prepared as follows:
The test article was weighed and transferred to a mortar. A small volume of vehicle (arachis oil) was added and mixed with a pestle to form a smooth paste. The mixture was transferred to the formulation bottle and the mortar and pestle rinsed with the vehicle, which was subsequently added (together with any remaining vehicle) to the formulation bottle to achieve the final volume. Formulations were then mixed using a Silverson until visibly homogenous.
Duration of treatment / exposure:
Day 1 (dosing) = 0 hours
Day 2 (dosing) = 24 hours
Day 3 (dosing) = 45 hours
Day 3 (necropsy) = 48 hours.
Frequency of treatment:
The test article, vehicle and positive control were given as three administrations, at 0, 24 and 45 hours. All animals were sampled at 48 hours.
Post exposure period:
None
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Remarks:
Vehicle control
Dose / conc.:
37.5 mg/kg bw/day
Remarks:
N-isopropyl-N'phenyl-p-phenylenediamine
Dose / conc.:
75 mg/kg bw/day
Remarks:
N-isopropyl-N'phenyl-p-phenylenediamine
Dose / conc.:
150 mg/kg bw/day
Remarks:
N-isopropyl-N'phenyl-p-phenylenediamine
Dose / conc.:
150 mg/kg bw/day
Remarks:
Positive control (EMS)
No. of animals per sex per dose:
Six males per dose (N-isopropyl-N'phenyl-p-phenylenediamine)
Six males (Vehicle control)
Three males (Positive control (EMS)
Control animals:
yes, concurrent vehicle
Positive control(s):
Three male rats received 150 mg/kg day EMS (positive control)

Examinations

Tissues and cell types examined:
Micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow and normochromatic erythrocytes (NCE).
Details of tissue and slide preparation:
Bone Marrow Sampling and Micronucleus Slide Preparation

The isolated femur was cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow was flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes. The samples were filtered through cellulose columns, containing 50 mg/mL equal mix of type 50 and α-cellulose. Once the majority of the 2 mL had passed through the column a further 4 mL of serum was added to the sample tubes and loaded onto the columns.

Once filtered, the bone marrow cells were pelleted by centrifugation (200 g, 5 minutes, 15-25°C) and the supernatant aspirated and discarded. A further 3 mL of foetal bovine serum was added to the tubes followed by gentle resuspension of the cell pellet. The cells were pelleted again (as described above) and the supernatant aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two/three uniquely labelled slides. A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were air-dried, then fixed for 10 minutes in absolute methanol and rinsed several times in distilled water. One or two slides per animal was immediately stained for 5 minutes in 12.5 µg/mL acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then dried and stored protected from light at room temperature prior to analysis. Unstained slides were air-dried and initially stored at <-10°C with desiccant. Once final results were confirmed the reserve slides were transferred to storage at room temperature.
Evaluation criteria:
Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 500 cells (PCE plus NCE) had been analysed for the Range-Finder and Genotoxicity animals. Then at least 4000 PCE/animal were examined for the presence of MN for Genotoxicity animals only.
The following criteria were used for analysis of slides:
1. Cells were of normal cell morphology
2. Areas where erythrocytes overlapped were ignored
3. A MN was round or oval in shape
4. A cell containing more than one MN was scored as a single micronucleated cell
5. MN that were refractive, improperly stained or not in the focal plane of the cell were judged to be artefacts and were not scored.
Statistics:
Data Evaluation
The experimental unit of exposure for in vivo studies is the animal, and all analysis was based on individual animal response.

Treatment of Micronucleus Data
After completion of microscopic analysis and decoding of the data the following were calculated:
1. %PCE for each animal and the mean for each group. The group mean %PCE values were examined to see if there was any decrease in groups of treated animals that could be taken as evidence of bone marrow toxicity
2. Frequency of MN PCE (i.e. MN per number of PCE scored) and %MN PCE for each animal and the group mean %MN PCE (standard deviation).
The numbers of MN PCE in the vehicle control animals were compared with the laboratory's historical control data to determine whether the assay was acceptable.
For each test article and vehicle control group, inter-individual variation in the numbers of MN PCE was estimated by means of a heterogeneity chi-square calculation. The numbers of MN PCE in each treated group were compared with the numbers in vehicle control groups by use of the Wilcoxon rank sum test. The tests were interpreted with one-sided risk for increased frequency with increasing dose. Probability values of p0.05 were accepted as significant. In addition the Terpstra-Jonckheere test for dose response was performed.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
No clinical signs of toxicity were observed at 37.5, 75 or 150 mg/kg/day. Macroscopically, there were no findings. Microscopically, in the liver, hepatocyte cytoplasmic alteration was present at 75 or 150 mg/kg/day. Stomach effect s were at 150 mg/kg/day.
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Micronucleus Analysis

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited group mean %PCE that were similar to the concurrent vehicle control group and which were within/comparable to the laboratory’s historical vehicle control data. There was no evidence of any test article-induced toxicity to the bone marrow (as would usually be indicated by a decrease in %PCE values compared to the vehicle control group).

Animals treated with N-isopropyl-N'phenyl-p-phenylenediamine at all doses exhibited MN PCE frequencies that were similar to the concurrent vehicle control group and that fell within the laboratory’s historical vehicle control data. There were no statistically significant increases in micronucleus frequency for any of the groups receiving the test article, compared to the concurrent vehicle control.

N-isopropyl-N'phenyl-p-phenylenediamine: Summary of Micronucleus Data

 Group / Dose Level

(mg/kg/day)

Total No. PCE Scored 

 Total No. of MN PCE

Mean %PCE 

%MN PCE

Mean

%MN PCE

Standard

Deviation

Heterogeneity

X²       

 Heterogeneity

Significance

 

 Wilcoxon

P value     

  Wilcoxon

Significance

 1M / Vehicle (0)

 24000

 43

 44.10

0.18 

 0.07

 5.71

 NS

 -

 -

 2M / N-isopropyl-N'phenyl-p-

phenylenediamine (37.5)

 24000

 28

 42.30

0.12 

 0.08

 9.73

 NS

 0 .9177

 NS

 3M / N-isopropyl-N'phenyl-p-

phenylenediamine (75)

 24000

 24

 42.07

 0.10

 0.09

 17.52

 P<0.01

 0.9199

 NS

 4M / N-isopropyl-N'phenyl-p-

phenylenediamine (150)

 24000

 28

 42.13

 0.12

 0.03

 1.57

NS 

 0.9437

 NS

 5M / EMS (150)

 12000

 186

 47.00

 1.55

 0.77

 

 

 0.0119

  P<0.05

Terpstra-Jonckheere trend test (Groups 1, 2, 3, 4; upper tail) P-value: 0.9236 NS

M        Male

MN        Micronucleus

PCE       Polychromatic erythrocyte

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, N-isopropyl-N'phenyl-p-phenylenediamine, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, when tested up to 150 mg/kg/day (an estimate of the maximum tolerated dose for this study). In conclusion, the test is negative.
Executive summary:

N-isopropyl-N'phenyl-p-phenylenediamine was tested for its potential to induce micronuclei (MN) in the polychromatic erythrocytes (PCE) of the bone marrow of treated rats.

Under the conditions of this study, N-isopropyl-N'phenyl-p-phenylenediamine, did not induce an increase in micronucleated polychromatic erythrocytes of the bone marrow, when tested up to 150 mg/kg/day (an estimate of the maximum tolerated dose for this study). In conclusion, the test is negative.