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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 December 2016 - 14 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No. of test material: 150726 (Test item No.: 16/0252-1)
- Expiration date of the lot/batch: 26 July 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany GmbH
- Females nulliparous and non-pregnant: yes
- Age at study initiation: males: 14 - 15 weeks, females: 13 weeks
- Housing: individually in polycarbonate cages
- Diet: ground Kliba maintenance diet mouse-rat “GLP” (supplied by Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: drinking water, ad libitum
- Acclimation period: 28 days

DETAILS OF FOOD AND WATER QUALITY:
- The food used in the study was assayed for chemical and microbiological contaminants.
- The drinking water is regularly assayed for chemical contaminants as well as for the presence of microorganisms.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 -24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% sodium carboxymethyl cellulose in drinking water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, 0.5% sodium carboxymethyl cellulose in drinking water was filled up to the desired volume and subsequently released with a magnetic stirrer. The test substance preparations were produced weekly, at least.

VEHICLE
- Concentration in vehicle: 1.25, 4.0, 12.5 g/100 mL
- Amount of vehicle: 10 mL/kg bw/d
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. At the beginning (during pre-mating), twice during gestation and once during lactation of the study each 3 samples were taken from the lowest and highest concentration for potential homogeneity analyses. These samples were used as a concentration control at the same time. At the time points mentioned above, one sample from the mid concentration was additionally taken for concentration control analysis. The samples collected at the beginning of the administration period and during the lactation period were analysed.
The stability of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water was demonstrated over a period of 7 days at room temperature. As the test substance preparations were not stored longer than this time period, the stability was guaranteed. The concentrations of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water were found to be in the range of 103-112% of the nominal concentration. These results demonstrated the correctness of the concentrations of the test substance in 0.5% sodium carboxymethyl cellulose in drinking water.
Duration of treatment / exposure:
The duration of treatment covered a 2-week premating period and mating in both sexes as well as entire gestation and lactation period in females up to one day prior to the day of schedule sacrifice of the animals (End of treatment: males: Day 28, females: Day 58 or 63)
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
125 mg/kg bw/day (nominal)
Remarks:
Considering a purity of the test substance plus water (whole product), the effective dose levels of the test substance itself was 100 mg/kg bw/d.
Dose / conc.:
400 mg/kg bw/day (nominal)
Remarks:
Considering a purity of the test substance plus water (whole product), the effective dose levels of the test substance itself was 300 mg/kg bw/d.
Dose / conc.:
1 250 mg/kg bw/day (nominal)
Remarks:
Considering a purity of the test substance plus water (whole product), the effective dose levels of the test substance itself was 1000 mg/kg bw/d.
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on dose range finding study

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations included: morbidity, pertinent behavioral changes and/or signs of overt toxicity

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
- Detailed clinical observations included: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos, assessment of the feces discharged during the examination (appearance/ consistency), assessment of the urine discharged during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: at study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning)

FOOD CONSUMPTION:
- Food consumption was determined.

FOOD EFFICIENCY:
- Food consumption was not determined.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: males: at study termination (Day 29), females: PND 14 (Day 50)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 parental animals per sex and group
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: males: at study termination (Day 29), females: PND 14 (Day 50)
- Animals fasted: Yes
- How many animals: 5 parental animals per sex and group
- Parameters checked in table 2 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males: Day 24, females: Day 55
- Dose groups that were examined: 5 parental animals per sex and group
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

THYROID HORMONES: Yes
- Time schedule for collection of blood: males: at study termination (Day 29), females: PND 14 (Day 50)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: Total thyroxine (T4)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)

HISTOPATHOLOGY: Yes (see table 4)
Other examinations:
ORGAN WEIGHTS: Yes (see table 5)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Soft feces were observed in 2 of 10 males and 6 of 10 females of test group 3 (1000 mg/kg bw/d), starting on pre-mating day 10 until mating in both sexes and in 1 of 10 females of test groups 2 (300 mg/kg bw/d) and 1 (100 mg/kg bw/d), observed on pre-mating day 13.
The finding also occurred during mating in all males and 6 of 10 females of test group 3, starting on mating day 1 in both sexes until mating day 14 in males and mating day 2 in females, respectively. In each 1 of 10 females of test groups 2 and 1, soft feces were observed between mating day 1 and 3 as well as 1 and 8, respectively. During post-mating, soft feces were still observed in all males of test group 3 until sacrifice.
Soft feces were also observed in all females of test group 3 (1000 mg/kg bw/d) between gestation days 0 and 21. It was also observed in 1 of 10 females of test group 2 (300 mg/kg bw/d) between gestation days 0 and 4.
This finding was considered to be related to treatment but not assessed as an adverse and toxicologically relevant effect.
Mortality:
no mortality observed
Description (incidence):
No animal died prematurely in the present study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test substance-related changes in mean body weights were observed for male and female animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Mean body weight change values were significantly decreased in male animals of test groups 3 (1000 mg/kg bw/d) and 2 (300 mg/kg bw/d) between pre-mating days 7 and 13.
As the changes occurred rather sporadically and no significant deviations occurred in mean body weights, they were assessed to be non-adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption values in female animals of test group 3 (1000 mg/kg bw/d) were significantly lower during pre-mating and significantly higher in females of test group 2 (300 mg/kg bw/d) on gestation day 20.
These values were still within a normal range typical for this strain of rats and, therefore, the deviations to the control were assessed to be without toxicological relevance.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No test substance-related changes in water consumption were observed.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes among hematological parameters were observed.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No treatment-related, adverse changes among clinical chemistry parameters were observed.
In female animals of test groups 2 and 3 (300 and 1000 mg/kg bw/d) alkaline phosphatase (ALP) activities were significantly higher compared to controls. The values were marginally above the historical control range (ALP 0.78-1.11 µkat/L). However, the ALP mean in test group 3 was only 43% higher compared to that one of the controls. Moreover, the ALP increase was the only clinical pathology alteration in these individuals. Therefore, this change was regarded as maybe treatment-related, but not adverse (ECETOC Technical Report No. 85, 2002).
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional observational battery:
Deviations from "zero values" were obtained in several animals. However, as most findings were equally distributed between test-substance treated groups and controls, without a dose-response relationship or occurred in single animals only, these observations were considered as incidental.
The following examinations were performed during FOB and are assessed individually:

Quantitative Parameters:
Rearing was significantly increased in female animals of test group 1 (100 mg/kg bw/d). As no dose-response relationship occurred, the change was assessed as being spontaneous in nature and not related to treatment.

Home cage observations:
No test substance-related effects were observed.

Open field observations:
Male animal No. 33 of test group 3 (1000 mg/kg bw/d) had soft feces.

Sensorimotor tests/reflexes:
No test substance-related effects were observed.

Motor activity measurement:
Regarding the overall motor activity, no test substance-related deviations were noted for male and female animals.
Comparing the single intervals with the control groups, significantly decreased value was measured for male animals of test group 1 (100 mg/kg bw/d) at interval 8. The difference was regarded to be incidental and not related to treatment as single interval was not changed in a dose-dependent manner and the overall motor activity was not affected.
No changes were observed for female animals in test groups 1, 2 and 3 (100, 300 and 1000 mg/kg bw/d) when compared to the control group.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
When compared to control group 0 (set to 100%), all mean absolute and relative weight parameters did not show significant differences.
Gross pathological findings:
no effects observed
Description (incidence and severity):
All findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related findings were observed in male and female animals.
All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.
The stages of spermatogenesis in the testes of males of the high dose test group were comparable to those of the controls. In the ovaries of control and high dose females the different stages of functional bodies (especially corpora lutea) were present and normal.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
No treatment-related findings were observed in male and female animals.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
1 250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: main constituent (corrected for purity, 100 % purity)
Sex:
male/female
Basis for effect level:
other: No test substance related adverse effects were observed.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this Combined Repeated Dose Toxicity Study with the
Reproduction/Developmental Toxicity Screening Test, the oral administration by gavage of
ACVA (4,4'-Azobis[4-cyanovaleric] acid) to Wistar rats revealed no signs of systemic toxicity
up to a dose level of 1000 mg/kg bw/d in animals of both sexes.
Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was 1000
mg/kg bw/d for male and female Wistar rats.
The NOAEL for reproductive performance and fertility was also set to 1000 mg/kg bw/d for
male and female Wistar rats.
The NOAEL for developmental toxicity was 1000 mg/kg bw/d.