diethyl pyridine-2,4-dicarboxylate

Repeated dose toxicity: dermal

Administrative data

Endpoint:

sub-chronic toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-03-01 to 2012-09-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

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, Portage, Michigan- Age at study initiation: at least 8 weeks- Weight at study initiation: 232-278 g males, 176-211 g females- Fasting period before study: no- Housing: individually in suspended, stainless steel, wire-mesh type cages- Diet (e.g. ad libitum): ad libitum- Water (e.g. ad libitum): ad libitum- Acclimation period: 14 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20-26.1- Humidity (%): 30-70- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Type of coverage:

open

Vehicle:

other: water/ethanol 1:1 (w/w)

Details on exposure:

TEST SITE- Area of exposure: dorsal surface on the left and on the right side- % coverage: 5 % of the total body surface area- Type of wrap if used: no- Time intervals for shavings or clipplings: as necessary approximately 14 to 20 hours prior to the next applicationREMOVAL OF TEST SUBSTANCE- Washing (if done): wiped with a Wypall®, wet with tap water- Time after start of exposure: following the 6 hour dosing periodTEST MATERIAL- Amount(s) applied (volume or weight with unit): 1.5 mL/kg- Concentration (if solution): 0, 167, 333, 500 mg/mL- Constant volume used: yesVEHICLE- Justification for use and choice of vehicle (if other than water): no dataUSE OF RESTRAINERS FOR PREVENTING INGESTION: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test solutions prepared for the study were evaluated for homogeneity (at 167 and 500 mg/mL) and concentration (at 0 and the three tested concentrations). Appropriate samples were collected while stirring using a positive displacement pipette, and placed into amber glass scintillation vials. All analytical work was conducted by MPI Research, Inc. The test solutions prepared for homogeneity analysis were determined to be homogeneous and accurately prepared with % relative standard deviations of 1.090 and 0.408 at the 167 and 500 mg/mL concentrations, respectively. The test solutions prepared during the study were found to be accurately prepared as the average % recoveries and % relative standard deviations at each concentration met the acceptability criteria.

Duration of treatment / exposure:

91 consecutive days

Frequency of treatment:

once daily for approximately 6 hours

Doses / concentrations

Remarks:

Doses / Concentrations:0, 250, 500, 750 mg/kg/dayBasis:nominal per unit body weight

No. of animals per sex per dose:

10 male and 10 female

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of a prior 2-week toxicity study performed by dermal route in rats at 500, 750, and 1000 mg/kg/day. The conditions of application were as follows: 6-hour non-occlusive application (rinsing after 6 hours), with a protective collar (to avoid any ingestion), always on the same area corresponding to 10 % body surface. Scabs were observed at the application site at the dose levels of 750 and 1000 mg/kg/day and dose-related erythema were noted from Day 4 of the study at all the tested doses. In order to reduce the local effects, the highest tested dose chosen that is anticipated to be well tolerated in this study was 750 mg/kg/day and the test article was applied on alternate sites of application.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: twice dailyDETAILED CLINICAL OBSERVATIONS: Yes- Time schedule: prior to randomization and weekly during the studyDERMAL IRRITATION (if dermal study): Yes- Time schedule for examinations: daily prior to dosingBODY WEIGHT: Yes- Time schedule for examinations: one day after receipt, prior to randomization and weekly during the studyFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: YesFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: NoWATER CONSUMPTION: NoOPHTHALMOSCOPIC EXAMINATION: Yes- Time schedule for examinations: pretest and prior to terminal necropsy (Day 90)- Dose groups that were examined: 0 and 750 mg/kg/day dose groupsHAEMATOLOGY: Yes- Time schedule for collection of blood: prior to terminal necropsy- Anaesthetic used for blood collection: Yes (carbon dioxide)- Animals fasted: Yes- How many animals: all animals- The following parameters were examined: leucocyte count (total and absolute differential), erythrocyte count, hemoglobin, hematocrit, mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (calculated), absolute reticulocytes, platelet count, blood cell morphology; Coagulation parameters: prothrombin time, activated partial thromboplastin time.CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: prior to terminal necropsy- Animals fasted: Yes- How many animals: all animals- The following parameters were examined: alkaline phosphatase, total bilirubin (with direct bilirubin if total bilirubin exceeds 1 mg/dL), aspartate aminotransferase, alanine aminotransferase, sorbitol dehydrogenase, urea nitrogen, creatinine, total protein, albumin, globulin and A/G (albumin/globulin) ratio (calculated), glucose, total cholesterol, triglycerides, electrolytes (sodium, potassium, chloride), calcium, phosphorus.URINALYSIS: Yes- Time schedule for collection of urine: at least 12 hours prior to terminal necropsy- Metabolism cages used for collection of urine: Yes- Animals fasted: Yes- The following parameters were examined: volume, color and appearance, specific gravity, pH, protein, glucose, bilirubin, ketones, blood, urobilinogen, microscopy of centrifuged sediment.NEUROBEHAVIOURAL EXAMINATION: Yes- Time schedule for examinations: once during Week 13 (Day 86)- Dose groups that were examined: all dose groups- Battery of functions tested: activity and arousal, posture, rearing, bizarre behavior, clonic and tonic movements, gait, mobility, stereotypy, righting reflex, response to different stimuli (approach, click, tail pinch, and touch), palpebral closure, pupil response, piloerection, exophthalmus, lacrimation, salivation, and respiration. The amount, qualitative and/or quantitative measures, of defecation and urination. Forelimb and hindlimb grip strength.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes: Adrenal (2)*, Aorta, Bone with marrow [femur], Bone with marrow [sternum], Bone marrow smear,, Brain [cerebrum, midbrain, cerebellum,medulla/pons]*, Epididymis (2)*, Eye including optic nerve (2), Galt [Gut Associated Lymphoid Tissue], Gastrointestinal tract: esophagus, stomach [glandular and nonglandular], duodenum, jejunum, ileum, cecum, colon, rectum; Gonads: ovary (2)* with oviduct (2), testis (2)*; Gross lesions, Heart*, Kidney (2)*, Lacrimal gland, exorbital (2), Liver [3 sections collected; 2 examined]*, Lung with bronchi [collected whole; 2 sections examined], Lymph nodes: mandibular and mesenteric, Mammary gland [only process females], Pancreas, Pituitary, Prostate and seminal vesicle (2), Salivary gland, mandibular/sublingual [2 collected; 1 examined], Salivary gland, parotid [2 collected; 1 examined], Sciatic nerve, Skeletal muscle, biceps femoris, Skin (normal and treated), Spinal cord [cervical, thoracic, and lumbar], Spleen*, Thymus*, Thyroid/parathyroid (2)*, Tongue, Trachea, Urinary bladder, Uterus [both horns]/Cervix, Vagina*Weighed organ(2) Paired organHISTOPATHOLOGY: Yes

Statistics:

For body weights, food consumption, hematology (except leukocyte counts), coagulation, clinical chemistry, organ weights, FOB (continuous endpoints) the group pair-wise comparisons (Levene's/ANOVA-Dunnett's/Welch's) were performed.For leukocyte counts the log transformation/group pair-wise comparisons were performed.For urinalysis (urine volume, specific gravity, pH) the rank transformation with Dunnett's test was performed.For dermal scoring and FOB (categorical endpoints) the Cochran Mantel Haenszel test was performed.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Dermal irritation:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITYAll animals survived to the scheduled terminal necropsy on Day 92.No significant clinical findings attributed to test article were noted during the study. During Weeks 9 to 13, unkempt appearance was observed in a male rat at 500 mg/kg/day, in three males at 750 mg/kg/day, and in one female at 750 mg/kg/day. Unkempt appearance is considered to be related to the test article, as it was not observed in animals in the control or 250 mg/kg/day groups. However, this effect is considered to be non-adverse based on the lack of other test article related findings.An abrasion on the thoracic region of male animal number 1028 at 500 mg/kg/day was observed during Weeks 9 to 13. The abrasion was near where the collar was applied during the 6-hour period and was not at the site of application. Therefore it was not considered to be related to test article administration.BODY WEIGHT AND WEIGHT GAINA very slight decrease in body weight gain was observed in treated males, but is not considered to be adverse due to the low magnitude of the change. No effect of test article administration was observed on female body weight.FOOD CONSUMPTIONNo effectsOPHTHALMOSCOPIC EXAMINATIONNo effect of test article administration was observed on ophthalmoscopic examinations. One male at 750 mg/kg/day was observed with chorioretinal hypoplasia in the left eye at the terminal examination, but is considered to be within limits of variation commonly encountered in animals of the sex, age, and strain.HAEMATOLOGYNo effectsCLINICAL CHEMISTRYIn both sexes at all dose levels there were mild test article-related decreases in cholesterol (up to 48 %) compared to controls. These changes followed a dose response but were not considered adverse or toxicologically relevant. There were also minimal and sporadic increases in phosphorus and/or chloride in both sexes at multiple dose levels that were not considered biologically or toxicologically relevant based on the absence of a dose response, their small magnitude and their inconsistent nature.There were frequent statistically significant alterations among albumin, globulin, and/or albumin/globulin ratio in both sexes at most dose levels that were considered likely to be indirectly test article-related. However, given their small magnitude, sporadic nature, and lack of a dose response, these findings were not considered biologically relevant.In treated females, a dose-related increase was observed in total bilirubin (10, 30, and 50 % at 250, 500, and 750 mg/kg/day respectively). In addition, one male and one female animal receiving 750 mg/kg/day (animal numbers 1078 and 1039) had mild to moderate increases in ALT, AST, and sorbitol dehydrogenase, compared to expected ranges. These findings are not likely to be test article-related based on their sporadic nature.URINALYSISNo significant and biologically relevant alterations attributed to test article were observed among urinalysis parameters in either sex at any dose level. There were occasional differences found in urine volume, pH and specific gravity that were considered nontoxicologically meaningful due to their sporadic nature and the inherent variability of these endpoints. There were also some variations between treatment groups among biochemical urinary components (occult blood, protein, etc.).NEUROBEHAVIOURNo effectsORGAN WEIGHTSNo effectsGROSS PATHOLOGYNo effectsHISTOPATHOLOGY: NON-NEOPLASTICTest article-related microscopic findings were present in males at 750 mg/kg/day and were limited to the non-glandular stomach (5 of 10 rats) and tongue (2 of 10 rats). Microscopic findings at the limiting ridge of the non-glandular stomach consisted of minimal erosion (4 of 10 rats) or ulcer (1 of 10 rats), minimal to mild hyperplasia of the epithelium, minimal to mild hyperkeratosis, and minimal mixed leukocyte infiltrates in the submucosa subjacent to the limiting ridge. Microscopic findings in the tongue consisted of minimal hyperkeratosis and minimal hyperplasia of the squamous epithelium of the tongue. Because of the absence of test article related findings at the treated dose sites and the presence of these findings in the tongue and non-glandular stomach in random animals in one sex, these microscopic findings may have been secondary to licking and subsequent unintended ingestion of the test article. Consequently, these microscopic findings were considered to be non-adverse.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The NOAEL is the highest tested dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Homogeneity of test solutions

Dose Level, mg/kg/day	Nominal Concentration, mg/mL	Average Calculated Concentration, mg/mL	Average Recovery^a,b, %	Relative Standard Deviation, %
250	167	161.2750	96.8	1.090
750	500	477.8709	95.6	0.408
^aAverage recovery (%) was calculated from the nominal concentration ^bThe acceptance criteria for recovery (%) is 100±5% of the nominal concentration.

 

Table 2. Concentration of test solutions

Dose Level, mg/kg/day	Nominal Concentration, mg/mL	Average Calculated Concentration, mg/mL	Average Recovery^a,b,c, %	Relative Standard Deviation^a, %
0	0.00	BLQ	NA	NA
250	167	158.9979 –175.5932	95.4 – 105.4	0.254 – 0.540
500	333	318.2704 – 352.2814	95.5 – 105.7	0.458 – 0.642
750	500	477.0787 – 534.0797	95.4 – 106.8	0.009 – 1.186
^aResults are the range of values determined during Weeks 1, 4, 8 and 13. ^bAverage recovery (%) was calculated from the nominal concentration ^cThe acceptance criteria for recovery (%) is 100±5% of the nominal concentration. BLQ – Below the Limit of Quantification (<0.0320 mg/mL) NA – Not Applicable

Applicant's summary and conclusion

Conclusions:

The once daily topical administration of Mexoryl SBU at 250, 500 and 750 mg/kg/day to rats at alternating application sites for 91 consecutive days did not produce any significant or adverse effects. The no-observed-adverse-effect-level (NOAEL) in rats is considered to be at least 750 mg/kg/day, the highest level tested.

Executive summary:

This study was conducted in order to evaluate the potential subchronic toxicity of the test article, Mexoryl SBU, in rats after 91 consecutive days of daily cutaneous application according to the OECD TG 411.

Three treatment groups of 10 male and 10 female Sprague-Dawley, CD® [Crl:CD®(SD)] rats were administered the test article at respective dose levels of 250, 500 and 750 mg/kg/day by dermal route for approximately 6 hours. During the 6 hour dosing period, the site was not occluded and each animal had an Elizabethan-type collar. One additional group of 10 animals/sex served as the control and received the vehicle, purified water/ethanol 1:1 (v/v) in the same conditions as the treated groups. The test article or vehicle was administered to all groups at a constant dose volume of 1.5 mL/kg to two alternating dorsal sites.

Observations for morbidity, mortality, injury and the availability of food and water were conducted twice daily for all animals. Detailed clinical observations on all animals were conducted weekly. Functional observational battery (FOB) evaluations were conducted on all animals once during Week 13 (Day 86). Dermal irritation scoring was conducted on all animals daily, prior to dosing. Ophthalmoscopic examinations were conducted on all animals during the acclimation period and on all animals at 0 and 750 mg/kg/day prior to terminal necropsy. Body weights and food consumption were measured and recorded weekly for all animals. Blood and urine samples for clinical pathology evaluations were collected from all animals prior to the terminal necropsy. At study termination, necropsy examinations were performed, organ weights were recorded, and tissues were microscopically examined.

All animals survived to the scheduled terminal necropsy on Day 92. No significant or adverse test article-related findings were observed on clinical observations, FOB evaluations, body weight or food consumption measurements, ophthalmoscopic examinations, hematology, coagulation, clinical chemistry, urinalysis, or macroscopic evaluations, or organ weight measurements.

Microscopic findings attributed to the administration of the test item were limited to the males at 750 mg/kg/day and were present in the non-glandular stomach and tongue. The limiting ridge of the non-glandular stomach had multiple findings in 5 of the 10 male rats at

750 mg/kg/day. These included minimal erosion or ulcer, minimal to mild hyperplasia of the epithelium, minimal to mild hyperkeratosis, and minimal mixed leukocyte infiltrates in the submucosa subjacent to the limiting ridge. There was minimal hyperkeratosis and minimal hyperplasia of the squamous epithelium of the tongue in 2 of 10 male rats at 750 mg/kg/day. Because of the absence of findings attributed to test article at the treated sites and because of the presence of these findings in the tongue and non-glandular stomach in limited number of animals in one sex only, these microscopic findings were considered to be consistent with a possible unintended oral exposure to the test article. Although, there is no documentation of any licking or oral exposure during the study, the potential existed for test article to be present within the cage by coming into contact with the application site following

administration and then ingested by the animal. Consequently, these microscopic findings were considered non-adverse.

The once daily topical administration of Mexoryl SBU at 250, 500 and 750 mg/kg/day to rats at alternating application sites, for 91 consecutive days did not produce any significant or adverse effects. The no-observed-adverse-effect-level (NOAEL) in rats is considered to be at least 750 mg/kg/day, the highest level tested.