diethyl pyridine-2,4-dicarboxylate

Genetic toxicity: in vivo

Administrative data

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:

20.10.2005 - 09.09.2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed under GLP and according to OECD guidelines

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Commission Directive No. 2000/32/EC, B12, 8 June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Mexoryl SBU

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

Mexoryl SBU

Constituent 2

Reference substance name:

diethyl pyridine-2,4-dicarboxylate

EC Number:

680-341-5

Cas Number:

41438-38-4

Molecular formula:

C11H13N04

IUPAC Name:

diethyl pyridine-2,4-dicarboxylate

Details on test material:

- Name of test material: MEXORYL SBU- The test material is the same than the one mentioned in section 7.2.1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories France, L'Arbresle, France.- Age at study initiation: 6 weeks- Weight at study initiation: males: 192.3 g (ranging from 164.1 to 232.1 g), females: 133.6 g (ranging from 118.0 to 143.6 g)- Assigned to test groups randomly: yes- Fasting period before study: No- Housing: Housed by groups in polycarbonate cages. Each cage contained autoclaved sawdust. - Diet (e.g. ad libitum): free access to A04 C pelleted maintenance diet- Water (e.g. ad libitum): ad libitum- Acclimation period: at least 5 days before the day of treatmentENVIRONMENTAL CONDITIONS- Temperature (°C): 22 ± 2- Humidity (%): 30 to 70- Air changes (per hr): at least 12 cycles/hour of filtered non-recycled fresh air- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

other: oral and subcutaneous

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% methylcellulose in purified water- Justification for choice of solvent/vehicle: not reported- Concentration of test material in vehicle: Experiment 1: 50, 100 and 200 mg/mL for the oral route, 400 mg/mL for the subcutaneous routeExperiment 2: 300, 350 and 400 mg/mL by subcutaneous route- Amount of vehicle (if gavage or dermal):Experiment 1: 10 mL/kg for the oral route, 5 mL/kg for the subcutaneous routeExperiment 2: 5 mg/L for the subcutaneous route- Lot/batch no. (if required): 014 K 00811

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test item was ground to a fine powder using a mortar and pestle in the vehicle.

Duration of treatment / exposure:

Preliminary toxicity tests: single treatmentExperiment 1: single treatmentExperiment 2: 1 or 2 administrations (separated by 24 h)

Frequency of treatment:

Preliminary toxicity tests: one treatmentExperiment 1: one treatmentExperiment 2: one treatment or two treatments separated by 24 hours

Post exposure period:

Preliminary toxicity tests: 48 h observation periodExperiment 1: bone marrow sampled 24 or 48 h after treatmentExperiment 2: bone marrow sampled 24 or 48 h after treatment

Doses / concentrations

Remarks:

Doses / Concentrations:Experiment 1, oral: 500, 1000 and 2000 mg/kg/day. Experiment 1, subcutaneous: 2000 mg/kg/day. Experiment 2, subcutaneous: 1500, 1750, 2000 mg/kg/day. Basis:nominal conc.

No. of animals per sex per dose:

- Preliminary toxicity test: 2000 mg/kg, oral, 1 administration, sampling after 24 h: 3 males, 3 females2000 mg/kg, subcutaneous, 1 administration, sampling after 24 h: 3 males, 3 females2000 mg/kg, subcutaneous, 2 administrations: 3 females- Experiment 1: Vehicle, oral, sampling after 24 h: 5 males, 5 femalesVehicle, oral, sampling after 48 h: 5 males, 5 females500 mg/kg, oral, sampling after 24 h: 5 males, 5 females1000 mg/kg, oral, sampling after 24 h: 5 males, 5 females2000 mg/kg, oral, sampling after 24 h: 5 males, 5 females2000 mg/kg, oral, sampling after 48 h: 5 males, 5 femalesVehicle, subcutaneous, sampling after 24 h: 5 males, 5 femalesVehicle, subcutaneous, sampling after 48 h: 5 males, 5 females2000 mg/kg, subcutaneous, sampling after 24 h: 5 males, 5 females2000 mg/kg, subcutaneous, sampling after 48 h: 5 males, 5 femalesPositive control, oral, sampling after 24 h: 5 males, 5 females- Experiment 2: Vehicle, subcutaneous, 2 administrations, sampling after 24 h: 5 females2000 mg/kg, subcutaneous, 2 administrations, sampling after 24 h: 5 femalesVehicle, subcutaneous, 1 administration, sampling after 24 h: 5 femalesVehicle, subcutaneous, 1 administration, sampling after 48 h: 5 females1500 mg/kg, subcutaneous, 1 administration, sampling after 24 h: 5 females1750 mg/kg, subcutaneous, 1 administration, sampling after 24 h: 5 females2000 mg/kg, subcutaneous, 1 administration, sampling after 24 h: 5 females2000 mg/kg, subcutaneous, 1 administration, sampling after 48 h: 5 femalesPositive control, oral, sampling after 24 h: 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide- Justification for choice of positive control(s): not reported- Route of administration: oral- Doses / concentrations: 15 mg/kg, one administration, 10 mL/kg

Examinations

Tissues and cell types examined:

- clinical signs and mortality- bone marrow cells: sampling at 24 or 48 hours after treatment

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:Since no toxic effects were observed at 2000 mg/kg in the preliminary experiment, the top dose-level was 2000 mg/kg/day for both the oral and subcutaneous routes. The two other selected dose-levels were 500 and 1000 mg/kg/day for the oral route in the first experiment and 1500 and1750 mg/kg/day for the subcutaneous route in the second experiment.DETAILS OF SLIDE PREPARATION:- animals sacrificed using CO2- removal of femur, bone marrow flushed and suspended in fetal calf serum- separation of anucleated erythrocytic cells from other myeloic cells using a cellulose column- centrifugation of the eluate containing the cells and removal of supernatant- spreading of a small drop of the re-suspended cell pellet on a slide- air drying of the smear and staining with Giemsa- coding of slides for blind scoringMETHOD OF ANALYSIS:- number of MPE (Micronucleated PE) counted in 2000 polychromatic erythrocytes- scoring of 1000 Erythrocytes to establish Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio- Experiment 1: microscopic examination of slides for each type of coloration (Giemsa and acridine orange due to equivocal results obtained with Giemsa)- Experiment 2: microscopic examination of slides using Giemsa coloration only

Evaluation criteria:

For a result to be considered positive, a statistically significant increase in the frequency of MPE must be demonstrated when compared to the concurrent vehicle control group. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of data obtained.

Statistics:

Normality and homogeneity of variances were tested using a Kolmogorov Smirnov test and a Bartlett test. If normality and homogeneity of variances were demonstrated, the statistical comparisons were performed using a Student t-test (two groups) or a one-way analysis of variance (≥ three groups) followed by a Dunnett test (if necessary). If normality or homogeneity of variances was not demonstrated, a Mann/Withney test (two groups) or a Kruskall Wallis test (≥ three groups) was performed followed by a Dunn test (if necessary). All these analyses were performed using the software SAS Enterprise Guide V2, with a level of significance of 0.05 for all tests.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY- Dose range: 2000 mg/kg- Solubility: not reported - Clinical signs of toxicity in test animals: Piloerection in 1/3 males and 3/3 females 5 hours after treatment following single administration by subcutaneous route. No mortality and no other clinical signs were noted. - Evidence of cytotoxicity in tissue analyzed: no tissue analyzed- Rationale for exposure: It is recommended in the guideline OECD 474 to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.- Harvest times: not done- High dose with and without activation: not applicable- Other: NoRESULTS OF DEFINITIVE STUDY- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): not applicable- Induction of micronuclei (for Micronucleus assay): Experiment 1, oral treatment, Giemsa analysis: No statistically significant increase in mean values of MPE compared to vehicle group in males and females at 24 h harvest time, but slight dose-related increase. Experiment 1, subcutaneous treatment, Giemsa analysis: No increase in MPE in males. Statistically significant increase in females at 24 h harvest time, no increase at 48 h harvest time. Experiment 1, oral treatment, Acridine orange analysis: Mean values of MPE equivalent to those of vehicle group.Experiment 1, subcutaneous treatment, Acridine orange analysis: Mean values of MPE equivalent to those of vehicle group.Experiment 2, subcutaneous treatment, Giemsa analysis: Statistically significant decrease in frequency of MPE in females given 2000 mg/kg. - Ratio of PCE/NCE (for Micronucleus assay):Experiment 1, oral treatment, Giemsa analysis: Slight statistically significant increase in PE/NE ratio in females given 2000 mg/kg at 48 h harvest timeExperiment 1, subcutaneous treatment, Giemsa analysis: PE/NE ratio equivalent to those of vehicle group. Experiment 1, oral treatment, Acridine orange analysis: Slight but statistically significant decrease in PE/NE ratio in females treated with 2000 mg/kg, considered as a sign of systemic toxicity Experiment 1, subcutaneous treatment, Acridine orange analysis: PE/NE ratio equivalent to those of vehicle groupExperiment 2, subcutaneous treatment, Giemsa analysis: Statistically significant decrease in PE/NE ratio in females given 2000 mg/kg twice. - Appropriateness of dose levels and route: only piloerection at the recommended upper limit of 2000 mg/kg- Statistical evaluation: See above

Any other information on results incl. tables

Experiment 1

In the view of the equivocal results obtained with Giemsa coloration in Experiment 1, an analysis of the available duplicates of the slides was undertaken using an acridine orange coloration. The use of a DNA-specific stain like acridine-orange may allow to check whether the results obtained with Giemsa stain were an artefact or not.

Plasma level of the test item

Since the test item MEXORYL SBU is a diester, it can be metabolized/hydrolyzed to give the corresponding monoesters (mixture of the monoesters) or diacide. Given the high levels of esterases in the body, in the blood and in the skin, the plasma levels of MEXORYL SBU was not determined. Moreover, given that blood samples of the first experiment were not protected with a anti-esterase agent (NaF), only the diacide form of the test item could be analysed in the plasma, while the monoester derivates were found in the plasma (protected with NaF) of the second experiment.

Results of the plasma analysis (μg/mL):

For both experiments, the analysis of the plasma showed that animals given MEXORYL SBU (a diester) at 2000 mg/kg/day by the oral (single administration) or subcutaneous route (one or two administrations) were exposed to the test item as significant amounts of either the monoester forms or the diacide form of the test item were found in the plasma

	Experiment 1		Experiment 2
Route (sampling time)	Oral (0.5h)	Subcutaneous (0.5h)	Subcutaneous (0.25h)	Subcutaneous (2h)
Plasma protection	None	None	NaF	NaF
MEXORYL SBU (diester)	ND	ND	ND	ND
monoesters	BLQ (M and F)	BLQ (M and F)	166±26 (F)	145±20 (F)
diacide	254±8 (M) 279±77 (F)	241±88 (M) 476±162 (F)	NA	NA

Experiment 1 : BLQ < 1.29 μg/mL

Experiment 2 : BLQ < 1.29 μg/mL

ND : not determined

NA : not analyzed

M : Male

F : Female

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negativeUnder the experimental conditions, the test item did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after administrations by oral or subcutaneous route, at dose-levels up to 2000 mg/kg/day.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce structural or numerical damage in bone marrow cells of rats. The study was performed in accordance with GLP and OECD TG 474.

Methods

A preliminary toxicity test was performed to define the dose-levels to be used for each experiment of the cytogenetic study.

In the first experiment

Three groups of five male and five female Sprague-Dawley rats received a single oral route administration of the test item at the dose-levels of 500, 1000 and 2000 mg/kg.

One group of five male and five female Sprague-Dawley rats received a single subcutaneous route administration of the test item at the dose-level of 2000 mg/kg.

One group of five males and five females received the vehicle (0.5% methylcellulose) by oral route under the same experimental conditions, and acted as control group for the oral route.

One group of five males and five females received the vehicle (0.5% methylcellulose) by subcutaneous route under the same experimental conditions, and acted as control group for the subcutaneous route.

One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 15 mg/kg.

In the second experiment

Three groups of five female Sprague-Dawley rats received by subcutaneous route a single administration of the test item at the dose-levels of 1500, 1750 and 2000 mg/kg and one group of five female Sprague-Dawley rats received subcutaneous route administration of the test item at the dose-level of 2000 mg/kg/day over a 2-day period.

One group of five females received the vehicle (0.5% methylcellulose) under the same experimental conditions (one or two treatments separated by 24 hours), and acted as control group.

One group of five females received the positive control test item (cyclophosphamide) once by

oral route at the dose-level of 15 mg/kg.

The animals given a single treatment of the test item and of the vehicle were killed 24 or 48 hours after treatment (first and second experiments), the animals given two treatments of the test item and of the vehicle control were killed 24 hours after the second treatment (second experiment) and the animals of the positive control group were killed 24 hours after treatment (both experiments). Bone marrow smears were then prepared. For each animal, the number of the Micronucleated Polychromatic Erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The Polychromatic (PE) and Normochromatic (NE) Erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

The slides of the first experiment were evaluated using Giemsa and acridine orange colorations. Those of the second experiment were evaluated using Giemsa coloration only.

Results

First experiment

No clinical signs and no mortality were observed in the animals of either sexes given 500, 1000 or 2000 mg/kg/day by oral route, or 2000 mg/kg/day by subcutaneous route.

Giemsa analysis

By the oral route, no statistically significant increase in the mean values of MPE was induced in the groups treated with the test item when compared to those of the vehicle group. However, a slight dose-related increase was noted in both males and females at the 24-hour harvest time.

At the 48-hour harvest time, a slight statistically significant increase in the PE/NE ratio was noted in females given 2000 mg/kg/day by oral route.

By the subcutaneous route, no noteworthy increase in the MPE was noted in males at the 24- or 48-hour harvest time. A slight but statistically significant increase in frequency of the MPE (p < 0.01) was noted in females at the 24-hour harvest time, while no noteworthy increase in the MPE was noted in females at the 48-hour harvest time.

In the view of these equivocal results (obtained with Giemsa coloration) and at the request of the Sponsor, an analysis of the available duplicates of the slides was undertaken using an acridine orange coloration. Indeed, there are several publications indicating that basophilic granules can stain and appear like micronucleus with Giemsa coloration. Even though a cellulose column was used to separate the anucleated erythritic cells from the other myeloic cells of the bone marrow, some granules released from disrupted mast cells might have passed through the column with the erythrocytes. The use of a DNA-specific stain like acridine-orange may allow to check whether the results obtained with Giemsa stain were an artefact or not.

Acridine orange analysis

A slight but statistically significant decrease in the PE/NE ratio was noted in females from the high-dose group, treated by oral route (0.5 ± 0.1 versus 1.0 ± 0.4 for the vehicle control group; p < 0.05). This decrease is considered as a sign of the systemic exposure of the animals to the test item. The mean values of PE/NE ratio in the other groups treated with the test item by oral and subcutaneous routes were equivalent to those of the vehicle group.

The mean values of MPE in the groups treated with the test item by the oral and subcutaneous routes were equivalent to those of the vehicle group. Moreover, by the oral route, the slight doserelated increase in the frequency of MPE noted in both sexes at the 24-hour harvest time with the Giemsa coloration was not confirmed with the acridine orange coloration.

Whatever the coloration used, cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The first experiment was therefore considered valid.

At the request of the Sponsor, in order to check the reliability of the increase in the frequency of MPE observed in females given the test item by subcutaneous route, a second experiment was performed using the same treatment schedule as the first experiment (a single treatment with 24-and 48-hour harvest times) with additional dose-levels in a close-range as well as a modified

treatment schedule (two treatments separated by 24 hours and a single 24-hour harvest time).

Second experiment

No clinical signs and no mortality were observed in the females given (by subcutaneous route) 1500, 1750 or 2000 mg/kg/day once, or 2000 mg/kg/day twice separated by 24 hours.

Whatever the treatment schedule, no statistically significant increase in the frequency of MPE was observed in the second experiment using the Giemsa coloration only.

At the 48-hour harvest time in females given 2000 mg/kg/day once, a statistically significant decrease in the frequency of MPE was noted.

In females given 2000 mg/kg/day twice (two treatments separated by 24 hours), a statistically significant decrease in the PE/NE ratio was noted.

Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The second experiment was therefore considered valid.

For both experiments, the analysis of the plasma showed that animals given Mexoryl SBU (a diester) at 2000 mg/kg/day by the oral (single administration) or subcutaneous route (one or two administrations) were exposed to the test item as significant amounts of either the monoester forms or the diacide form of the test item were found in the plasma.

Discussion

In the first experiment, the slight statistically significant increase in the frequency of MPE observed using the Giemsa coloration (females given 2000 mg/kg/day once by subcutaneous route) was not observed with the slides of the same experiment but colored using acridine orange (a DNA-specific coloration). Moreover, no statistically significant increase in the frequency of MPE was noted in the second experiment using either a closer range of dose-levels with a single administration, or a modified treatment schedule (two administrations separated by 24 hours). Consequently, the slight statistically significant increase in the frequency of MPE noted in the first experiment was not considered as biologically relevant.

Conclusion

Under our experimental conditions, the test item Mexoryl SBU (batch No. 002 D 001) did not induce damage to the chromosomes or the mitotic apparatus of rat bone marrow cells after administrations by oral or subcutaneous route, at dose-levels up to 2000 mg/kg/day.