Registration Dossier

Administrative data

Description of key information

Subacute combined repeated dose and reproduction / developmental screening study (OECD 421): NOAEL for parental animals = 100 mg/kg/day.

Subchronic  toxicity study: 13-weeks oral (Gavage) toxicity study (from 1986) with  NA-11 in rats, similar to OECD Guideline 408 EU Method B.26: NOEL = 500 mg/kg/day for both sexes based on food intake suppression.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
Deviations:
no
Qualifier:
according to
Guideline:
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar (Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 326 gr (males) or 210 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

IN-LIFE DATES
From: 31 January to 22 March 2013
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase according to a validated method (Project 206595). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. 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:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42 to 50 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation (up to the day prior to scheduled necropsy). Two Group 1 Females, one Group 2 Female and one Group 4 Female were not dosed during littering.
Frequency of treatment:
Once daily, 7 d/w
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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 thirteen weeks toxicity study (Yokosuka Project No. T-448) in which 200, 500 and 1500 mg/kg were tested in Wistar rats. Based on the findings in this 90-day toxicity study blood sampling for haematology, clinical biochemistry and weight determination of the liver and kidneys were added to the repro screening study.
Positive control:
Not required.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals.
The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY: yes

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

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: between 7.00 and 10.30 a.m.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
No
Sacrifice and pathology:
GROSS PATHOLOGY
- All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- All animals: According to test guidelines plus collection of liver and kidneys.

ORGAN WEIGHTS
- All animals: Epididymides, kidneys, liver and testes.


HISTOPATHOLOGY
- According to test guidelines plus liver.
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 (many-to-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.

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:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, clinical signs of toxicity were noted mainly for females during the observation period. These signs consisted of hunched posture (five females), rales (two females and one male), piloerection (nine females), pale faeces (three females), and lean appearance (two females).
Mortality:
mortality observed, treatment-related
Description (incidence):
At 1000 mg/kg, one Group 4 female was found dead on Day 4 post-coitum. This animal showed hunched posture, piloerection and a lean appearance during the five days before its death. She showed a body weight loss of 15% from Day 1 of mating until Day 0 post-coitum (over a 7 day period). No cause of death could be established.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, body weights and body weight gain were decreased for both sexes.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, food consumption before or after allowance for body weight was decreased for females during the complete treatment period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, several clinical biochemistry parameters were considered to be affected by treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg for females, relative liver weights were slightly increased.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg, there were test item-related macroscopic findings in female rats. Findings consisted of emaciation noted for two females and pale discolouration of the liver (correlating to hepatocellular vacuolation) in one female.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic findings were noted in the liver of female rats treated at 300 mg/kg and in both sexes treated at 1000 mg/kg.
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
At 1000 mg/kg, one Group 4 female was found dead on Day 4 post-coitum. This animal showed hunched posture, piloerection and a lean appearance during the five days before its death. She showed a body weight loss of 15% from Day 1 of mating until Day 0 post-coitum (over a 7 day period). No cause of death could be established.

All remaining animals survived their scheduled study period.

CLINICAL SIGNS
At 1000 mg/kg, clinical signs of toxicity were noted mainly for females during the observation period. These signs consisted of hunched posture (five females), rales (two females and one male), piloerection (nine females), pale faeces (three females), and lean appearance (two females).

Salivation seen after dosing among animals of the 300 and 1000 mg/kg dose group 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).

Incidental findings that were noted included chromodacryorrhoea, alopecia and ptosis. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS
At 1000 mg/kg, body weights and body weight gain were decreased for both sexes.
For mean body weights this was statistically significant on Days 15 and 29 of study for the males and on Day 20 post-coitum for the females. A statistically significantly decrease was noted for mean body weight gain during the complete treatment period for the males, and for the females on Day 1 of mating and Day 20 post-coitum.
It should be noted that one Group 4 male was most affected; showing a slight body weight loss over the study period, and that one Group 4 female showed a reduced body weight gain during post-coitum as she had implantation sites only.

No toxicologically relevant changes in body weights were noted at 100 and 300 mg/kg.
The statistically significantly decreased body weight gain on Day 8 of treatment for the males was not considered toxicologically relevant as the change was slight and recovered during treatment.

FOOD CONSUMPTION
At 1000 mg/kg, food consumption before or after allowance for body weight was decreased for females during the complete treatment period. This was statistically significant from Day 7 post-coitum onwards.

During the first week of treatment, food consumption was also decreased for both sexes at 100 and 300 mg/kg. However, as this recovered during the remainder of the treatment period it was not considered toxicologically significant.
The statistically significantly findings during post-coitum at 100 and 300 mg/kg were not considered toxicologically relevant as the changes were slight and inconsistent over time.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

Any statistically significant changes (reticulocytes at 1000 mg/kg, prothrombin time at 100 and 300 mg/kg, activated partial thromboplastin time and platelets at 300 mg/kg) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

CLINICAL BIOCHEMISTRY
At 1000 mg/kg, several clinical biochemistry parameters were considered to be affected by treatment. For both sexes, these changes consisted of increased mean concentrations of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase (for males, the increased average of the first two parameters were caused by one Group 4 animal). In addition for females, levels of total protein were decreased and total bilirubin and urea were increased.

The remaining statistically significant changes (total protein, creatinine and calcium at 300 mg/kg, cholesterol at 100 and 1000 mg/kg, and chloride at 100 and 300 mg/kg) were not considered toxicological significant as changes occurred in the absence of a dose-related trend and remained within normal limits.

MACROSCOPIC EXAMINATION
At 1000 mg/kg, there were test item-related macroscopic findings in female rats. Findings consisted of emaciation noted for two females and pale discolouration of the liver (correlating to hepatocellular vacuolation) in one female.

A soft yellowish nodule/focus at the epididymides was noted for two males/group at 100, 300 and 1000 mg/kg. At histopathological examination, in five cases (one control and two high dose) it showed to be a granuloma. These findings were within normal limits for this type of study, and were not considered treatment related.

The incidence of other incidental 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 dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance.

ORGAN WEIGHTS
At 1000 mg/kg for females, relative liver weights were slightly increased. In addition, terminal body weights were decreased for males at 300 and 1000 mg/kg and for females at 1000 mg/kg.

Statistically significant changes for the male liver at 300 mg/kg and male kidneys at 1000 mg/kg were considered not to be a sign of toxicity as they were due to relatively high control values or lower terminal body weight and/or no treatment-related distribution was noted.

Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

MICROSCOPIC EXAMINATION
Test item-related microscopic findings were noted in the liver of female rats treated at 300 mg/kg and in both sexes treated at 1000 mg/kg.

Findings in the periportal area:
• Oval cell and/or bile duct hyperplasia in 2/10 females (minimal) at 300 mg/kg and 6/10 females (up to moderate).
• Peribiliary lymphocytic inflammation in 2/10 females (up to slight) at 300 mg/kg and 5/10 females (up to slight) at 1000 mg/kg.
• Periportal hepatocellular hypertrophy in 1/10 females (minimal) at 300 mg/kg and 4/10 females (up to slight) at 1000 mg/kg.
• Increased incidence and severity of microvesicular and/or macrovesicular hepatocellular vacuolation in 6/10 females (up to moderate) at 1000 mg/kg compared to minimal degree in 1/10 males at 100 mg/kg and 1/10 females at 300 mg/kg.
• Increased number of mitosis (with bizarre mitosis) in 1/10 females at 1000 mg/kg.
• Hepatocellular cytoplasmic alteration at moderate degree in 1/10 males at 1000 mg/kg.

Other findings in the liver:
• Increased incidence and severity of single cell necrosis (mainly periportal and midzonal) up to moderate degree in 4/10 females at 300 mg/kg and 5/10 females at 1000 mg/kg, compared to minimal degree in 2/10 in control males, 1/10 males and 1/10 females at 100 mg/kg.
• Increased severity and/or incidence of centrilobular hepatocellular hypertrophy up to slight degree in 7/10 males and minimal degree in 5/10 females at 1000 mg/kg compared to minimal degree in 1/10 control males and 1/10 control females, 1/10 males at 100 mg/kg and 1/10 males at 300 mg/kg.

The remainder of the microscopic findings recorded were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

Reproductive performance:

There were four pairs treated at 1000 mg/kg that failed to sire or deliver healthy offspring and were therefore selected for histopathological examination of the reproductive organs. At microscopic evaluation of the uterus, implantation sites were noted for on Group 4 female. The remaining females that were not pregnant had histologically a normal cyclic reproductive tract. No cause for the failure to sire or deliver healthy offspring could be established from the sections examined.

Spermatogenic staging profiles were normal for all males examined.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
> 100 - < 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Key result
Dose descriptor:
NOAEL
Remarks:
Parental generation
Effect level:
> 300 - < 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
300 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Relevant for humans:
not specified

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%). No test substance was detected in the Group 1 formulation.  

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

Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Conclusions:
In this reproduction / developmental toxicity screening test on ADK STAB NA-11 by oral gavage in Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg/d toxic effects were observed in the parental animals, primarily in females. Parental NOAELs were derived at 100 mg/kg/d for females and at 300 mg/kg/d for males.
With regard to reproduction toxicity the NOAEL was derived at 300 mg/kg/d, for developmental toxicity it was derived at 100 mg/kg/d. However, based on the maternal toxicity observed at dose levels of 300 and 1000 mg/kg/d and the fact that the design of this study (OECD 421) is for screening for reproductive and developmental toxicity the results are considered inconclusive. For deciding on a respective classification (or not) a two-generation reproduction toxicity study (OECD 416) was performed.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
System:
hepatobiliary
Organ:
liver

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

In the recently performed combined repeated dose and reproduction / developmental screening study a NOAEL for parental animals was derived at the dose of 100 mg/kg/day. This value will be used in the CSA.

The main effect observed in the 13 week repeated dose toxicity study (from 1986) with NA-11 was suppression of food intake in the high dose groups (1500 mg/kg b.w./day), mainly in males. This effect is thought to be due to the large dosing volume of 15 ml/kg body weight. Other observed effects were considered as secondary effects of the reduced food intake. The NOEL was derived at the dose of 500 mg/kg/day.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

In the recently performed combined repeated dose and reproduction / developmental screening study significant signs of toxicity were observed leading to a parental NOAEL of 100 mg/kg/day. This NOAEL is used in the CSA. In the subchronic study performed in 1986 only mild effects of NA-11 were observed leading to a NOEL of 500 mg/kg/day.

Justification for classification or non-classification

Based on the NOAEL of 100 mg/kg bw/day derived from the recently performed combined repeated dose and reproduction / developmental screening study , NA-11 does not need to be classified regarding repeated dose toxicity according to REGULATION (EC) 1272/2008.