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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:
30 August 2012 - 30 October 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:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. QA statement)
Remarks:
Research Toxicology Centre S.p.A., Via Tito Speri, 12/14, 00040 Pomezia (Rome), ITALY
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
EC Number:
401-280-0
EC Name:
1-(N,N-bis(2-ethylhexyl)aminomethyl)-1,2,4-triazole
Cas Number:
91273-04-0
Molecular formula:
C19 H38 N4
IUPAC Name:
bis(2-ethylhexyl)[(1H-1,2,4-triazol-1-yl)methyl]amine
Details on test material:
- Physical state: Yellowish clear liquid
- Analytical purity: 97.7g/100g (quantitative 1H-NMR spectroscopy)
- Storage condition of test material: Room temperature

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Glx/BRL/Han)IGSBR
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: 7 to 8 weeks
- Weight at study initiation: 205 to 212 g for males and 172 to 190 g for females
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulphone solid bottomed cages measuring approximately 59.5x38x20 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week. During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42x26x18 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were before mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring approxiamtely 43x26x18 cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and changed at least 3 times a week.
- Diet: laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), ad libitum
- Water: ad libitum via bottles
- Acclimation period: 22 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 2°C
- Humidity (%): 40 to 70%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% CMC (Carboxymethylcellulose) + 0.1% polysorbate 80 in purified water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test substance was suspended in the vehicle. The formulations were prepared daily (concentrations of 2, 6 and 20 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Details on mating procedure:
Mating was monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
Female no. 61 (100 mg/kg bw/day) was paired with male no. 62 (100 mg/kg) for a total of 14 days. The pairing combination was then changed and the female was paired with another male of the same group (male no. 64, 100 mg/kg). Mating occurred with the second male.
Female no. 39 (10 mg/kg bw/day) was paired with male no. 40 (10 mg/kg) for a total of 14 days. The pairing combination was changed and the female was paired with another male of the same group (male no. 28, 10 mg/kg). Mating occurred with the second male indicated.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content check). Results of the analyses were within the limits of acceptance.
The stability was found to be 24 hours at room temperature in the concentration range from 2.0 to 20 mg/mL.
Samples of the formulations prepared on Week 1 and last Week were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 93430; BASF Project
No. 10Y0349/12X214), in the range from 2.0 to 20 mg/mL. Results of formulation analyses were within the acceptability limits.
Duration of treatment / exposure:
Males:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter through the day before necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weights on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
30 mg/kg bw/day (actual dose received)
Dose / conc.:
100 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 had been selected based on data obtained after 4-week administration of the test compound. Marked gastric irritation was observed in this study macroscopically and histologically at the high dose (200 mg/kg bw/day). Submucosal edema was observed in the stomach of one animal at 60 mg/kg bw/day. Based on these results, the dose level of 100 mg/kg bw/day was selected as high dose.
- Other: The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight

Examinations

Parental animals: Observations and examinations:
MORTALITY
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

CAGE SIDE OBSERVATIONS: Yes
All clinical signs were recorded for individual animals.
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions.

BODY WEIGHT: Yes
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0, 7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.

FOOD CONSUMPTION
Food consumption was recorded at weekly intervals whenever possible, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).

PARTURITION
A parturition check was performed from Day 20 to Day 25 post coitum. Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Litter observations:
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified.
Live pups were individually weighed on Days 1 and 4 post partum.
Observation was performed once daily for all litters.
Postmortem examinations (parental animals):
SACRIFICE
Female nos. 73 and 77 (both 100 mg/kg), sacrificed for humane reasons and those that had completed the scheduled test period, as well as all males, were killed with carbon dioxide.
Parental males: Males were sacrificed after the mating of all females, after 42/43 days of treatment. Female nos. 13, 19 (both from control group) and 21 (10 mg/kg) which did not litter were sacrificed on gestation Day 26.
Parental females: The females with live pups were sacrificed on Day 4 post partum.

GROSS NECROPSY
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
All females were examined also for the following:
a) number of visible implantation sites (pregnant animals);
b) number of corpora lutea

HISTOPATHOLOGY / ORGAN WEIGHTS
From all animals completing the scheduled test period, the organs indicated in table 1 were dissected free of fat and weighed. The ratios of organ weight to terminal body weight were calculated for each animal.
Samples of all the tissues listed in table 1 were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).
The tissues required for histopathological examination are listed in table 1. After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed. In the first instance the examination was restricted as detailed below:
a) Tissues specified in section 4.4.6 from all animals in the control and high dose group killed at term.
b) Tissues specified in section 4.4.6 from all animals killed or dying during the treatment period.
c) All abnormalities in all groups.
Postmortem examinations (offspring):
SACRIFICE
Pups were sacrificed by intraperitoneal injection of Sodium Thiopental on Day 4 post partum.

GROSS NECROPSY
All pups found dead in the cage were examined for external and internal abnormalities. All live pups were killed on Day 4 post partum and examined for external abnormalities and sex confirmation by gonadal inspection.
Statistics:
Group mean values were calculated for all parameters. Data from females which did not mate, with total resorption or non-pregnant and from dams without live young were excluded from group mean calculations as considered appropriate by the Study Director.
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05.
Reproductive indices:
Copulatory Index, Fertility Index, Pre- coital Interval, Pre-implantation loss, Pre-birth loss
Offspring viability indices:
Pup loss at birth, Cumulative pup loss on Day 4, Sex ratios

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
The only relevant signs were described in females no. 73 and 77 sacrificed for humane reasons. Salivation was observed in high dose animals sporadically in males and females at approximately 10-15 minutes post dosing on Week 4 of treatment.
Mortality:
no mortality observed
Description (incidence):
Two females of the high dose group receiving 100 mg/kg bw/day were sacrificed for humane reasons during the study. Female no. 77 was sacrificed due to difficulty in parturition on gestation Day 22. In addition, pallor and piloerection were noted at the despatch to necropsy. An abnormally enlarged placenta (18x16x11mm, located near the cervix) was observed in the uterus at necropsy and may have been the cause of difficulty in parturition. Since this animal was inconspicuous regarding clinical signs and body weight gain before, this finding was considered to be incidental.

An additional female (animal no. 73) was sacrificed for humane reasons on gestation Day 23. The signs at the despatch were: piloerection, liquid faeces, brown staining on muzzle, hunched posture and cold to touch. At necropsy the only sign described was brown staining on muzzle. A compound-related effect was not established, since these findings emerged at the day of sacrifice, and the animal appeared normal before (i.e. there were no signs indicating toxicity based on clinical signs or body weights).
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Means of body weight were comparable between control and treated groups both in males and females throughout the study.

No relevance was attributed to the slight increase in body weight gain noted on Week 1 at statistical analysis, in mid- dose males receiving 30 mg/kg bw/day, since the data refer to the interval allocation-dosing before the start of treatment.

The slight reduction in body weight gain noted on gestation Day 7, at statistical analysis, in high dose females receiving 100 mg/kg bw/day was considered to be a normal fluctuation and not related to treatment.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption was unaffected by treatment in both sexes during the study. No toxicological relevance was attributed to the lower food consumption observed in high dose females receiving 100 mg/kg bw/day on Day 7 of gestation.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were noted.

Histopathological evaluation of female no. 73 did not establish the reasons for the illness status of this animal. Marked haemorrhage of the placenta was observed in female no. 77.

The observed lesions had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Wistar Hannover rats of the same age, under our experimental conditions. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted

Reproductive function / performance (P0)

Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Unilateral implantation with total resorption was observed in one low dose female receiving 30 mg/kg bw/day.

Two females of the control group were not pregnant. All treated females proved to be pregnant.

The number of females with live pups on Day 4 post partum was: 8 in the control, 9 in the low dose group (10 mg/kg bw/day), 10 in the mid-dose group (30 mg/kg bw/day) and 8 in the high dose group (100 mg/kg bw/day).
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.

Gestation periods were similar in treated groups and controls. All pregnant dams gave birth between Days 22 and 23 post coitum.

Corpora lutea, implantations, total litter size, pre-implantation loss and pre-birth loss were similar between groups.

Details on results (P0)

.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
Parental toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
Fertility/Reproductive Toxicity
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No differences in total and live litter size, mean pup weight and sex ratio were noted between groups at birth or on Day 4 post partum.

At the daily general litter observations, individual pups of control and treated groups were smaller than others without dose relationship. This finding was not considered of toxicological significance for the following reasons: it is a normal condition in a litter that the pup weights are not homogeneous and some pups appear smaller than others. In addition, the observation was not confirmed by statistical significance in group mean data of litter weights or pup weights.

Three pups from 2 litters of the low dose group, all pups from 1 litter of the mid-dose group and all pups from 4 litters of the high dose group were found cold to touch at birth (Day 0 shortly after birth) or occasionally after birth without dose relationship. All findings were considered of no toxicological significance since litter data including pup cumulative loss were comparable between the control and the treated groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The findings noted at necropsy were similar between groups.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: highest dose tested
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) was considered to be 100 mg/kg bw/day for both sexes.

The NOAEL for reproductive and developmental toxicity was considered to be 100 mg/kg bw/day for both sexes.
Executive summary:

A GLP-compliant reproduction / developmental toxicity screening test was conducted in Wistar rats following OECD guideline 421. The purpose of this study was to provide information on toxic effects on rats of both sexes after repeated dosing, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, gestation, parturition and early lactation. The vehicle was 0.5% CMC (Carboxymethylcellulose) + 0.1% polysorbate 80 in purified water. All doses (0, 10, 30, 100 mg/kg bw) were administered at a constant volume of 5 mL/kg body weight to groups of 10 male and 10 female animals. Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 42/43 days. During the in-life phase, body weight, body weight gain, clinical signs, food consumption and mating performance were evaluated. At necropsy a detailed external and internal examination was performed. The histopathological examination, including identification of the stages of the spermatogenic cycle (males), was carried out in control and high dose groups. No treatment-related changes were seen. No signs of toxicological significance were observed at the clinical examination, body weights and food consumption. Fertility and copulatory index were unaffected by treatment. Terminal body weight and organ weights were unaffected by treatment. Two high dose females were sacrificed for humane reasons during late gestation. A compound-related effect was not established. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum. During the in-life phase, body weight, body weight gain, clinical signs, food consumption, oestrous cycle and mating performance were evaluated. The histopathological examination was carried out in females of control and high dose groups. No treatment-related changes were seen. Clinical signs of pups, as well as necropsy examination of pups sacrificed at termination or unscheduled deaths, were recorded. At necropsy a detailed external and internal examination was performed. Litter data, sex ratios and gestation length were recorded. No compound-related effects were noted during the in life phase, including mating, gestation, birth, nursing behaviour, and postnatal development of the offspring. Terminal body weight and organ weights were unaffected by treatment. Based on the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) was considered to be 100 mg/kg bw/day for both sexes. The NOAEL for reproductive and developmental toxicity was considered to be 100 mg/kg bw/day for both sexes.