2,2'-azobis[2-methylpropionamidine] dihydrochloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10.07.-25.09.2012

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:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 421

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

EC Number:

221-070-0

EC Name:

2,2'-azobis[2-methylpropionamidine] dihydrochloride

Cas Number:

2997-92-4

Molecular formula:

C8H18N6.2ClH

IUPAC Name:

2,2'-diazene-1,2-diylbis(2-methylpropanimidamide) dihydrochloride

Constituent 2

Reference substance name:

2,2'-azobis(2-amidinopropane)-dihydrochloride

IUPAC Name:

2,2'-azobis(2-amidinopropane)-dihydrochloride

Test material form:

other: granules

Details on test material:

- Name of test material (as cited in study report): 2,2'-azobis(2-amidinopropane)-dihydrochloride
- Substance type: organic
- Physical state: solid
- Analytical purity: 99.1 %
- Lot/batch No.: I59596
- Expiration date of the lot/batch: 31. Dec. 2012
- Storage condition of test material: ambient temperature, dark

Test animals

Species:

rat

Strain:

other: Wistar Han (IGS)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Duration of treatment / exposure:

Males were dosed daily for 43 - 50 days including the scheduled termination of the in-life phase. This included two weeks of dosing prior to mating and continued throughout the mating period until approximately four weeks post-mating.
Females were dosed two weeks prior to mating, covering at least two complete oestrous cycles, the variable time to conception, the duration of pregnancy and at least four days after delivery, up to and including the day before scheduled termination of the in-life phase. Therefore the duration of the study following acclimatisation depended on the female performance and was at least 48 days:
14 days pre-mating, up to 14 days until mating, an average of 21 days of gestation, and between 9 and 12 days of lactation.

Frequency of treatment:

daily

No. of animals per sex per dose:

12 animals per sex per dose

Control animals:

yes

Results and discussion

Results: maternal animals

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Effect levels (fetuses)

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Regarding the reproduction and development parameters gathered in this study no changes were obsered in all experimental groups that were definitely test item related.

During first and second mating phase, no apparent differences occured regarding the evidence of copulation and the number of females that achieved pregnancy. On two occasions, couples were separated due to an increase body weight of the female animals although no evidence for copulation was detected. All other females conceived within the first five days. Pregnancy lasted 21 days, 22 days, or 23 days. Four female animals of the high dose group delivered one or two stillbirth, as well as two females of the low dose group and three femals of the vehicle control group. None stillbirth was deformed or had developmental delay.

Two animals gave birth to their litters on day 18 of gestation phase. The high dose animal gave birth to one stillbirth. All other pups were of normal size and appearance, thus it could be assumed that sperm plug on day one or two of the mating phase was missed (couples were separated without sperm plug on day 9 or 7 of the mating phase.

No difference between the dose groups were detectable regarding the number of dams with both, live young born at day zero (d0, day of birth and alive pups at day four (d4) of lactation. The mean numbers per dam of corpora lutea, implantations and live pups at d0 and d4 as well as the mean litter weight at d0 and d4 were normal for Wistar rats, and no statistically significant differences between the dose group were found. Stillbirth (n=11) were distributed rather homogenously among all dose groups.

On day 4pp one pup of an animal of the medium dose group had lost its right leg from the knee downwards. Apart from that, the pup was in a good condition until the end of the in-life phase. As the animal was unobtrusive on day 0pp it could be assumed that an injury was casual for the loss. None of the other animals had pups showing abnormalities that could be related to the test item.

The loss of offspring (pre-implantation, pre-natal or post-natal) were normal for primiparae rats of this strain and age. Although slightly

higher mean values regarding the pre-implantation losses could be observed for the test item treated animals when compared to the vehicle control animals, no test item related tendency could be observed.

No further abnormalities were detected during gestation and lactation phase. In summary, no differences were observed regarding reproduction success (achievement of pregnancy after indication of copulation, litter size and survival of pups) for all test item related animals when compared to the vehicle control animals.

Applicant's summary and conclusion

Conclusions:

The parental NOAEL of development toxicity was considered to be 60 mg/kg/day for male and female rats.

Executive summary:

In the present study oral uptake of the test item at a dose of 60 mg/kg body weight did not induce toxic effects on the development and the reproduction of Wistar rats.

Mild discomfort throughout the whole application period was observed for the animals treated with the high and the medium dose of the test item (salivation after application, blending of mucous membranes at nose and mouth, respirators sound) . A biological and particularly toxicological relevance of those observation could not be substantiated due to the sporatic incidence of the symptoms.

In comparison to the vehicle control group significant decreased body weight values were determined for the male animals treated with the high dose of the test item during week 4 whereas no conspicuities regarding body weight were observed for the male animals treated with the medium and the low dose. Similarly, no significant differences were observed between all female test item treated animals and the respective vehicle control animals.

A tendency of slightly enhanced amounts for water consumption within the test item treated male animals could observed throughout the whole in-life phse whereas no differences were detected for the female animals. An impact of the test item administration on the food consumption could not be observed, neither for male nor for female animals.

The most prevalent findings during necrospy were swollen lymph nodes. All other findings were observed with low incidences and without any test item related tendency. They were thus regarded to be spontaneous in nature.

Histopathological examination of the ovaries as well as of the epididymides/testes did not produce any evidence ot pathomorphological findings that are considered to be due to a toxic effect of the test item.

Regarding the reproduction and development parameters gatherd in this stud no changes were observed that were definitely treatment related. reproduction success (achievment of pregnancy after the indication of copulation, litter size and survival rate of pups) was comparable between test item related animals and the vehicle control group.

A daily oral administration of the test item to male Wister rats at dose level of 6,7 mg, 20 mg and 60 mg/kg body weight over a time period of 43 to 50 days did not produce any pathological evidence to toxic effects on the reproduction performance of male rats. However, effects of the spermatogenesis may not have had an adequate time to become evident (such as reduced sperm counts affecting the fertility) as chemical exposure does not cover a complete cycle of spermatogenesis in male test animals.

A daily oral administration of the test item to female Wistar rats at dose levels of 6,7 mg, 20 mg and 60 mg/kg body weight over a time period of 48 to 68 days did not produce any pathological evidence for toxic effects on reproduction preformance of female rats.

With respect to the results the NOAEL of the test item under the study conditons was estimated to be 60 mg/kg body weight. So far, no adverse effect has been observed regarding the reproduction of female and male rats. Nonetheless, due to the selectivity of the end points and the short duration of the study the data ascertained in this study do not indicate absolute safety with respect to the reproduction and development.