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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2011 - October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE, Experimental Toxicology end Ecology, 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): N-(3-Aminopropyl) imidazol
- Physical state: liquid / colorless, clear
- Analytical purity: 99.6 %
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: (P) 11-13 wks
- Weight at study initiation: (P) Males: 293-321 g; Females: 201-226 g
- Housing: individually in Makrolon type M III cages; exceptions: male and female partners were housed together during overnight matings, pregnant animals and their litters were housed together until PND 4
- Diet (ad libitum): groudn Kliba maintenance diet mouse/rat "GLP meal, Provimi Klibi SA, Kaiseraugust, CH
- Water (ad libitum): in water bottles
- Acclimation period: 6 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:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- The test substance was weighed in a graduated measuring flask depending on the dose group, topped up with highly deionized water and intensely mixed by shaking until completely dissolved.
- Volume administered each day: 10 ml/kg bw
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: overnight (about 04:00 p.m. until 07:00 - 09:00 a.m.)
- Proof of pregnancy: [sperm in vaginal smear] referred to as GD0 = day 0 of pregnancy
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
once during the study period
Duration of treatment / exposure:
- females: 14 days before mating, during pregnancy, until PND4
- males: 28 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 40, 200, 1000 mg/kg bw/d
Basis:
nominal in water
No. of animals per sex per dose:
10 per sex and group
Control animals:
yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes, at least daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data

BODY WEIGHT: Yes, weekly

FOOD CONSUMPTION: Yes, weekly, but not determined during mating period of F0 animals

WATER CONSUMPTION: No
Oestrous cyclicity (parental animals):
-
Sperm parameters (parental animals):
-
Litter observations:
PARAMETERS EXAMINED in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality up to PND4, clinical observation, presence of gross anomalies, weight gain PND1-4

GROSS EXAMINATION OF DEAD PUPS:
[yes, for external and internal abnormalities]
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [the next day after 28 days of exposure]
- Maternal animals: All surviving animals [at PND4]

GROSS NECROPSY
- Gross necropsy, special attention was given to the reproductive organs

HISTOPATHOLOGY / ORGAN WEIGHTS
- Weights in all animals: anesthetized animals, epididymes, testes
- Fixation in 4% formaldehyde or in modified Davidson´s solution: all gross lesions, cervix, coagulating gland, epididymes, ovaries, oviducts, prostate gland, seminal vesicles, testes, vagina, uterus
- Histopathology: Testes, epididymes, ovaries of control and high dose group
Postmortem examinations (offspring):
SACRIFICE: on PND4
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
GROSS NECROPSY: external examination and eviceration, macroscopic assessment of organs
Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding.
Statistics:
DUNNETT-test, FISHER´s EXACT test, WILCOXON-test, KRUSKAL-WALLIS test
Reproductive indices:
- Male mating index (%), male fertility index
- Female mating index (%), female fertility index (%), gestation index (%), live birth index (%), postimplantation loss (%)


Females: determination of the number of implantations and calculation of the postimplantation loss
Offspring viability indices:
- Viability index (%), sex ratio

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

There were no test substance-related adverse findings in any of the groups.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male animal (#32) of the high-dose group was found dead in study week 3 without any abnormal clinical or pathological findings. Therefore, this was considered to be incidential and not treatment-related.
Some animals of the high-dose group showed transient salivation, perstisting only for some minutes after daily dosing.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight of the high-dose males (up to -6% during study week 1 and 2) and food consumption of the high-dose males and females (-21% or -19% during study week 1) were statistically significantly reduced. Later on, both parameters were comparable to the control.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
- Male/female mating index = 100% (control, low-, mid-, high-dose). One low- and one mid-dose female (#117, 121) were not detected as being sperm positive. However, both had pups, giving evidence for successful copulation.
- Male/female fertility index = 100 % (control), 90% (low-, mid-dose), 80 % (high-dose). This is in the normal range of biological variation, inherent in the used rat strain. The infertile males (#17, 25, 34, 37) showed no relevant gross lesions. Minimal changes in testicles and epididymes were observed in male #34, but not severe enough to explain infertility. The non-pregnant femalse (#115, 125, 134, 137) had no relevant gross lesions.
- Mean duration of gestation: similar in all groups (22.0 - 22.5 d)
- Gestation index = 100 % in all groups
- Mean numer of implantation sites: comparable between control, low-, mid-, high-dose groups (13.5, 12.4, 13.0, 12.1)
- Post-implantation loss: comparable between control, low-, mid-, high-dose groups (10.7%, 7.7%, 6.7%, 7.4%)
- Mean number of F1 pups, delivered per dam: comparable between control, low-, mid-, high-dose groups (12.0, 11.4, 12.0, 11.3)
- Live birth index: 100% (control, low-dose), 99% (mid-dose), 94%* (p<= 0.05, high-dose); mainly caused by dam #138 with 4/10 stillborn pups.
- Number of stillborn pups: statistically increased in high-dose group (0, 0, 1, 5*); mainly caused by dam #138 with 4/10 stillborn pups. However, all respective values were in the historical control range and thus relect the normal range of biological variation inherent in the used rat strain.

ORGAN WEIGHTS (PARENTAL ANIMALS)
All mean absolute and relative weight parameters did not show significant differences compared to the control.

GROSS PATHOLOGY (PARENTAL ANIMALS)
All findings occured either indivdually or were biologically equally distributed over all groups and were not considered treatment-related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No findings were observed in the high-dose group, that could explain the lack of offspring in males (#34, 37) or lack of pregnancy in females (#134, 137). Minimal changes observed in the testicles (multifocal tubular degeneration) and epididymes (cell debris) of male #34 frequently occur as background lesions in male rats without impairing fertility. These changes were not considered treatment-related. All other findings occured either indivdually or were biologically equally distributed over all groups and were not considered treatment-related.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance-related adverse findings at any dose

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

Details on results (F1)

There were no test substance-related adverse findings in any of the groups.

VIABILITY (OFFSPRING)
- Mean number of F1 pups, delivered per dam: comparable between control, low-, mid-, high-dose groups (12.0, 11.4, 12.0, 11.3)
- Live birth index: 100% (control, low-dose), 99% (mid-dose), 94%* (p<= 0.05, high-dose); mainly caused by dam #138 with 4/10 stillborn pups.
- Number of stillborn pups: statistically increased in high-dose group (0, 0, 1, 5*); mainly caused by dam #138 with 4/10 stillborn pups. However, all respective values were in the historical control range and thus relect the normal range of biological variation inherent in the used rat strain.
- Viablilty index during lactation (PND0-4): no test substance related changes between control, low-, mid-, high-dose (99, 93%*, 99, 96%). The slightly reduced index in the low-dose group resulted of the accidental death of 6 pups, which were placed with the wrong dam after weighing and immediatly killed).
- Sex distribution and ratios did not show substational differences between the groups.

CLINICAL SIGNS (OFFSPRING)
No test substance-related adverse were observed.

BODY WEIGHT (OFFSPRING)
Mean pup bw and pup bw change of all groups were statistically comparable.
One female runt each was seen in the control and the high-dose group, one male runt in the mid-dose group.

GROSS PATHOLOGY (OFFSPRING)
- One F1 pup showed a spontaneous finding (dilated renal pelvis). There were no further findings.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance-related findings at any dose

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

In conclusion, under the conditions of this study the NOAEL for general systemix toxicity, reproductive toxicity performance and fertility is 1000 mg/kg bw/d for the F0 parental rats. Furthermore, the NOAEL for developmental toxicity was also 1000 mg/kg bw/d.

Applicant's summary and conclusion

Conclusions:
In conclusion, under the conditions of this OECD421 study the NOAEL for general systemix toxicity, reproductive toxicity performance and fertility is 1000 mg/kg bw/d for the F0 parental rats. Furthermore, the NOAEL for developmental toxicity was also 1000 mg/kg bw/d. Therefore, Aminopropyl-imidazol is considered to be non-toxic to reproduction (neither fertility, nor developmental toxicity) in this study.
Executive summary:

In a GLP compliant OECD421 study, N-(3-Aminopropyl)-imidazole was given daily to 10 Wistar rats per sex and group per gavage at doses of 0, 40, 200 and 1000 mg/kg bw/d (BASF SE, 2012) to screen fo potential reproductive and developmental toxicity. The exposure lasted for at least 28 days in males and for at least 38 days in females (2 weeks prior to mating, during the mating period of max. 2 weeks, about 1 week post-mating for males and for the entire gestation period up to PND 4 in females). The examination of clinical signs and mortality, of food consumption, body weight and organ weights, the gross pathology, histopathology and the reproductive performance relvealed no toxicologically relevant adveres effects in parental animals up to 1000 mg/kg bw/d. Furthermore, no toxicologically relevant developmental differences in the F1 pups were detected during this study considering viability, clincial signs, body weight and gross pathology of the F1 pups. Therefore, under the conditions of this study, the NOAEL for reproductive toxicity as well as for developmental toxicity in the F1 offspring is 1000 mg/kg bw/d.