Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
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

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: P males: 14 - 15 weeks, P 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

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 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
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight, for a maximum of 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually
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
Details on study schedule:
- F1 parental animals were not mated.
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 no treatment
Details on study design:
- Dose selection rationale: based on dose range finding study

Examinations

Parental animals: Observations and examinations:
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.

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily

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.

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

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)
Oestrous cyclicity (parental animals):
For all females of the pool estrous cycle normality was evaluated before the beginning of the administration.
In all parental females in the premating phase, estrous cycle length and normality was evaluated by preparing vaginal smears during a minimum of 2 weeks prior to premating, mating and throughout cohabitation until there is evidence of sperm in the vaginal smear.
Additionally, on the day of scheduled sacrifice, the estrous status was also determined in all female F0 rats.
Sperm parameters (parental animals):
Parameters examined in P male parental generations:[testis weight, epididymis weight
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

THYROID HORMONES: Yes
- Time schedule for collection of blood: PND 4 and 13
- Anaesthetic used for blood collection: Yes (isoflurane)
- How many animals: all surplus pups or 2 preferably female pups per litter at PND 4 as well as one male and one female pup per litter at PND 13
- Parameters checked: Total thyroxine (T4)
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals at Day 29
- Maternal animals: All surviving animals at Day 59 or 64

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues indicated in Table 4 were prepared for microscopic examination. The tissues indicated in Table 5 were weighed.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 13 days of age.
- These animals were subjected to postmortem examinations (external and macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
Statistics:
See Table 6.
Reproductive indices:
Mating index, fertility index, gestation index, live birth index, and postimplantation loss were calculated.
Offspring viability indices:
Viability index, survival index and sex ratio were calculated.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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
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.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Estrous cycle data revealed regular cycles in the rearing F1 females of all test groups including the control. The mean estrous cycle duration in the different test groups was 3.9 days in test groups 0 (control), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 3.8 days in test group 1 (100 mg/kg bw/d).
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
Male mating index
The male mating index calculated after the mating period for F1 litter was 100% in test groups 0 (control), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% in test group 1 (100 mg/kg bw/d).

Male fertility index
Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Male animal No. 5 of test group 0 (control), which was mated with female No. 105, male animal No. 7 of test group 0, which was mated with female animal No. 107, and male animal No. 120 of test group 1 (100 mg/kg bw/d), which was mated with female animal No. 120, did not generate F1 pups. Thus, the male fertility index was 80.0% in test group 0 (control), 90% in test group 1 (100 mg/kg bw/d) and 100% in test groups 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d).
These values reflected the normal range of biological variation inherent in the strain of rats used for this study-

Female mating index
The female mating index calculated after the mating period for F1 litter was 100% in test groups 0 (control; 0 mg/kg bw/d), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) and 90% in test group 1 (100 mg/kg bw/d).
The mean duration until sperm was detected (GD 0) was 2.5 days for test group 0, 3.3 days for test group 1, 4.0 days for test group 2 and 2.2 days for test group 3. These values reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Female fertility index
Most sperm positive rats delivered pups with the exception of female animal No. 105, which was mated with male animal No. 5 of test group 0 (control) and female animal No. 107, which was mated with male animal No. 7 of test group 0, had sperm in vaginal smear but delivered no pups and showed no implants. Thus, the female fertility index was 80% in test group 0 and 100% in test groups 1 (100 mg/kg bw/d), 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d). These values reflected the normal range of biological variation inherent in the strain of rats.

Duration of gestation
The mean duration of gestation was similar in all test groups, i.e. 22.2 days (test group 0), 22.1 days (test group 1), 22.4 days (test group 2) and 22.3 days (test group 3). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.

Gestation index
The gestation index was 100% in all test groups.

Effect levels (P0)

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 (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
All F1 pups of any test group (0-3) did not show adverse clinical signs up to scheduled sacrifice on PND 4 or PND 13.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The viability index indicating pup mortality during PND 0-4 was 100.0% in test group 0 (control) and test group 1 (100 mg/kg bw/d), 97.6% in test group 2 (300 mg/kg bw/d) and 99.1% in test group 3 (1000 mg/kg bw/d). The rate of liveborn pups in all test groups was not affected by the test substance, as indicated by live birth indices of 100%.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group. Each one female runt was seen in 2 litters of test group 0 (control group; 0 mg/kg bw/d) and one female runt was seen in each 1 litter of test groups 1 (100 mg/kg bw/d) and 3 (1000 mg/kg bw/d) on PND 1. All values were within the range of the biological variation inherent in the strain of rats used for this study.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance-related findings were observed.
Histopathological findings:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones:
No treatment-related alterations of T4 levels were observed.

Anogenital distance:
Anogenital distance and anogenital index were not affected in all F1 pups.

Nipple/areola anlagen:
The apparent number and percentage of male pups having areolae was not influenced by the test substance when examined on PND 13.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

open allclose all
Key result
Dose descriptor:
NOAEL
Generation:
F1
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
Generation:
F1
Effect level:
1 000 mg/kg bw/day
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 (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 1: Male mating indices after the mating periof for F1 litter

 

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Male mating
index [%]

100.0

90.0

100.0

100.0

Table 2: Female mating indices after the mating periof for F1 litter

 

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Female mating
index [%]

100.0

90.0

100.0

100.0

Table 1: Male fertility indices for F0 males

 

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Male fertility
index [%]

80.0

90.0

100.0

100.0

*p<0.05;**p<0.01

Table 2: Fertility indices for F0 females

 

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Female fertility
index [%]

80.0

100.0

100.0

100.0

*p<0.05;**p<0.01

Table 3: Sex ratio of live F1 pups

PND 0

Test group 0
(0 mg/kg bw/d)

Test group 1
(100 mg/kg bw/d)

Test group 2
(300 mg/kg bw/d)

Test group 3
(1000 mg/kg bw/d)

Live males [%]

49.5

54.1

53.7

57.8

Live females [%]

50.5

45.9

46.3

42.2

PND 13

 

 

 

 

Live males [%]

51.6

55.6

53.8

52.5

Live females [%]

48.4

44.4

46.2

47.5

 

Gestation index: The gestation index was 100% in all test groups.

Live birth indices: The rate live birth indices were 100% in all test groups.

Postimplantation loss: The postimplantation loss was 7.3% in test group 0 (control), 9.6% in test group 1 (100 mg/kg bw/d), 5.5% in test group 2 (300 mg/kg bw/d) and 5.3% in test group 3 (1000 mg/kg bw/d). These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. A treatment-related increase in postimplantation loss was not observed in any test group.

.

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.