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Description of key information

In a 90-day oral toxicity study with rats no toxicologically relevant effects up to and including the highest dose tested were observed, and therfore the NOAEL for FDCA is at least 1000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
november 2012 - march 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2012 by MHLW (0331 No.7), METI (No. 5) and MOE (No. 110331009)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Rat: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males 132-169 grams, females 113-144 grams
- Fasting period before study: no
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type) with sterilized sawdust as bedding material and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA) without cage-enrichment or bedding material.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals had no access to food.
- Water: Free access to tap water except during motor activity measurements.
- Acclimation period: At least 5 days before the start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 18 December 2012 To: 20 March 2013
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing between Days 1-10, and on a weekly basis from Day 11 onwards (based on results of stability analyses). Formulations were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information provided by the Sponsor.
- Concentration in vehicle: 1% Aqueous carboxymethyl cellulose
- Amount of vehicle (if gavage): 5 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on three occasions during the treatment phase, according to a validated method (project 496926). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions and over 8 days stored in the refrigerator was also determined (highest and lowest concentration, in Week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 days per week.
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on results of a 14-day oral range finding study with FDCA by daily gavage in the rat (project 501480).
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily. The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes, weely
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY: no

WATER CONSUMPTION: Yes
-Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest and at week 13
- Dose groups that were examined: pretest all animals, week 13 groups 1 and 4 (since no treatment-related ophthalmologic findings were noted in week 13, the eyes of the rats of Groups 2 and 3 were not examined).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight, for a maximum of 24 hours)
- How many animals: all
- Parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes (overnight, for a maximum of 24 hours)
- How many animals: all
- Parameters according to guidelines were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: during week 12 of treatment
- Dose groups that were examined: all
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength, locomotor activity

OTHER: organ weights of adrenal glands, spleen, brain, testes, epididymides, thymus, heart, uterus (including cervix), kidneys, prostate, liver, seminal vesicles including coagulating glands, ovaries, thyroid including parathyroid
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, according to guideline
HISTOPATHOLOGY: Yes, according to guideline
Statistics:
The following statistical methods were used to analyze the data:
-If the variables could be assumed to follow a normal distribution, the Dunnett-test (manyto- one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
-The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
-The Fisher Exact-test was applied to frequency data.
-The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
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 specified
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study period that was considered to be related to treatment with the test substance. One male at 1000 mg/kg bw/day was found dead on Day 82. No cause of death could be established histopathologically, nor did this animal or any other animal of the same dose group show any signs of impaired health condition. Therefore, this death was considered to be incidental in nature and unrelated to treatment with the test substance. One male at 1000 mg/kg bw/day and one female at 100 mg/kg bw/day died at blood sampling on the day of necropsy. Since neither of these two animals showed any signs of imminent death, they were considered to be related to the blood sampling procedure.
No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among all animals at 1000 mg/kg bw/day during the greater part of the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to taste of the test substance.

BODY WEIGHT AND WEIGHT GAIN
No toxicologically significant changes in body weights and body weight gain were noted. Males at 1000 mg/kg bw/day showed a lower body weight (gain) during the second half of treatment, achieving a level of statistical significance on several occasions. Means however remained within the range considered normal for rats of this age and strain, and therefore this change was considered to be of no toxicological relevance.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control
animals.

OPHTHALMOSCOPIC EXAMINATION
No toxicologically significant ophthalmology findings were noted. Incidental ophthalmology findings at pretest and/or in Week 13 consisted of focal corneal opacity, focal corneal oedema, and haemorrhage from the hyaloid vessel. The nature and incidence of these findings was within the range considered to be normal for rats of this age and strain, and was therefore considered to be of no toxicological relevance.

HAEMATOLOGY
No toxicologically relevant changes occurred in haematological parameters. The statistically significantly lower relative eosinophil counts of males at 300 and 1000 mg/kg bw/day remained with the range considered normal for rats of this age and strain and showed no clear doserelated trend. One male at 1000 mg/kg bw/day showed a high red blood cell distribution width (RDW). Other animals of the same group showed a red blood cell distribution width that was comparable to control values. No toxicological significance was therefore ascribed to these changes.

CLINICAL CHEMISTRY
No toxicologically relevant changes occurred in clinical biochemistry parameters. Any statistically significant changes in clinical biochemistry parameters were considered to be of no toxicological significance as they remained (essentially) within the range considered normal for rats of this age and strain, and occurred in the absence of a dose-related trend and supportive toxicologically relevant microscopic lesions. These changes consisted of higher aspartate aminotransferase activity (ASAT) in three males at 1000 mg/kg bw/day, lower total protein and cholesterol level in males at 1000 mg/kg bw/day, higher potassium level in males at 300 and 1000 mg/kg bw/day, higher sodium level in males at 100, 300 and 1000 mg/kg bw/day and lower inorganic phosphate levels in females at 100, 300 and 1000 mg/kg bw/day.

NEUROBEHAVIOUR
Pupillary reflex, static righting reflex and grip strength were normal in all animals, and no toxicologically relevant changes in hearing ability and motor activity were noted. One female at 1000 mg/kg bw/day showed absence of a hearing response. Since none of the other animals at this dose showed a similar finding, this was considered to be incidental in nature and to be of no toxicological relevance. Females at 300 and 1000 mg/kg bw/day showed a statistically significantly lower motor activity (for both total movements and ambulations). These changes did however not show a dose-related trend, means remained within the range considered normal for rats of this age and strain, and no supportive clinical signs were noted. Also, all groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period. Therefore, these variations were considered not to be of toxicological relevance.

ORGAN WEIGHTS
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. Any statistically significant changes in organ weights or organ to body weight ratios were considered not to be of toxicological relevance since they occurred in the absence of a dose-related trend, means remained within the range considered normal for rats of this age and strain, and/or were due to the lower terminal body weights (high dose males). These changes consisted of lower heart and prostate weight in males at 1000 and 100 mg/kg bw/day respectively, higher brain, thyroid and spleen to body weight ratio of males at 1000 mg/kg bw/day, and higher testes and epididymides to body weight ratio of males at 300 mg/kg bw/day.

GROSS PATHOLOGY
Necropsy did not reveal any toxicologically relevant alterations. The incidence of necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a doserelated incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

HISTOPATHOLOGY: NON-NEOPLASTIC
No toxicologically relevant microscopic findings were recorded. The slight increase in incidence/severity of vacuolation of the zona glomerulosa of the adrenal gland in one female at 300 mg/kg bw/day and in 6/10 males and 3/10 females at 1000 mg/kg bw/day can be regarded as adaptive in nature and was within physiological limits. This finding was therefore considered a nonadverse treatment related finding. All other microscopic findings were also within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Critical effects observed:
not specified

Accuracy of preparation.

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). A small response at the retention time of the test substance was observed in the chromatograms of the Group 1 formulation prepared for use in Week 1, Week 6 and Week 13. There is no analytical explanation for this response.Maximum contribution to the other samples was 1.5% based on peak area.

Homogeneity

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability

Formulations at the entire range were stable when stored at room temperature for at least 6 hours under normal laboratory light conditions and when stored in a refrigerator for at least 8 days.

Conclusions:
In a 90-day oral toxicity study with rats no toxicologically relevant effects up to and including the highest dose tested were observed, and therfore the NOAEL for FDCA is at least 1000 mg/kg bw/day.
Executive summary:

Wistar rats were treated with FDCA for 13 weeks by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day according to OECD test guidelines. Formulation analyses confirmed that formulations of test substance in 1% aqueous carboxymethyl cellulose were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature and at least 8 days in the refrigerator. No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). From the results presented in this report a NOAEL for FDCA of at least 1000 mg/kg bw/day was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
good

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Wistar rats were treated with FDCA for 13 weeks by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day according to OECD test guidelines. Formulation analyses confirmed that formulations of test substance in 1% aqueous carboxymethyl cellulose were prepared accurately and homogenously, and were stable over at least 6 hours at room temperature and at least 8 days in the refrigerator. No toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, ophthalmoscopy, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). From the results presented in this report a NOAEL for FDCA of at least 1000 mg/kg bw/day was established.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable study which has been performed according to OECD and/or EC guidelines and according to GLP principles.

Justification for classification or non-classification

Based on the absence of toxicologically relevant effects up to and including the highest dose tested (1000 mg/kg bw/day), FDCA does not need not to be classified according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.