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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September-November 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and reported study under GLP
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

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)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Nitrilotriacetonitrile
EC Number:
230-804-9
EC Name:
Nitrilotriacetonitrile
Cas Number:
7327-60-8
Molecular formula:
C6H6N4
IUPAC Name:
2-[bis(cyanomethyl)amino]acetonitrile
Details on test material:
Batch no.: CFC 9161
Expiry date: 02 June 2012
Purity: 99.6 +/- 1.5%
Water: 0.4 +/- 0.2%

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
See further

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
Dose formulations of the test material were prepared approximately every 2 weeks as suspensions in a vehicle of corn oil at graded concentrations of 0.5, 5.0 and 22.5 mg/ml, which corresponded to target doses of 1, 10 and 45 mg/kg/day, when administered at a dosing volume of 2 ml/kg. Dose levels of the test material were selected by the Sponsor based on the results of a 14-day range-finding study (Experimur Study No.: 09-570). Test material formulations of NTAN were previously shown to be stable for a minimum of 4 weeks when stored at room temperature. Test material formulations were stored at room temperature (approximately 22 ± 3 degrees C), and thoroughly mixed on a stir plate prior to and during dosing.
Details on mating procedure:
One male was housed with one female of the same dose level for up to 2 weeks. Daily vaginal smears, taken from the female rats during the mating period, were evaluated for the presence of sperm as well as the stage of the estrus cycle. The presence of sperm in the smear or a sperm-plug indicated a positive mating, and that day was designated as gestation day 0. Ten (10) spermpositive female rats per group were selected to produce the F1 generation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test material formulations were collected from the first preparation (i.e., prior to dosing, week 0), mid-way through the study (approximately week 3), and towards the end of the study (week 4), and analyzed for concentration. Samples for homogeneity (homogeneity is performed on duplicate samples collected from the top, middle and bottom of the test material formulation) were collected from the low and high dose formulations of the 1st preparation and analyzed concurrently with the first concentration analyses. A validated gas chromatography (GC) with flame ionization detection (FID) method was used to determine concentration and homogeneity of the NTAN dose formulations. The analytically-determined concentrations met the acceptance criterion of ± 15% of the target concentration as specified in the study protocol. Samples for homogeneity were collected from the low and high dose formulations at study initiation and were found to be within the protocol-specified acceptance criteria of ± 15%.
Duration of treatment / exposure:
2 weeks prior to mating, during mating, and until sacrifice (males: 31 doses; females: a minimum of 48 doses)
Frequency of treatment:
Daily
Details on study schedule:
See above
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 10, 45 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex main study, 5 additional rats/sex in the control and high dose groups (recovery animals kept for 14 days after the last treatment; these animals were not mated)
Control animals:
yes, concurrent vehicle
Positive control:
Not used

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION: Yes
WATER CONSUMPTION: No

see for furthet details and outcome, the repeated dose toxicity study (28-day).
Oestrous cyclicity (parental animals):
Daily vaginal smears were collected over 3 days prior to cohabitation to ensure and evaluate cyclicity. Vaginal smears were collected during the mating period until the presence of sperm was detected.
Sperm parameters (parental animals):
No
Litter observations:
Physical Examination of Rat Pups: Rat pups (F1 generation) were sexed and given a gross external physical examination on postnatal day 0 and again on postnatal day 4. Dead or stillborn pups were examined for gross external anomalies, and, when feasible (for fresh un-autolyzed pups), a visceral examination was performed.
Postmortem examinations (parental animals):
All animals, including those found dead, received a complete necropsy. Necropsy included examination of the external surface of the body, all orifices, the cranial, thoracic, and peritoneal cavities, and their contents. The uterus of each rat was examined for evidence of implantation sites and the number of sites was recorded; in addition, the corpora lutea were counted. Rats scheduled for necropsy were fasted overnight and were euthanized using sodium pentobarbital. Seevral tissues were collected. In addition, a bone marrow smear was collected and fixed for possible future evaluation. Testes and epididymides were fixed in Bouin’s solution, and the eyes and associated tissues (e.g., optic nerve and Harderian gland) were fixed in Davidson’s solution; all other tissues were fixed in
10% neutral buffered formalin.
Postmortem examinations (offspring):
Only in case of dead or stillborn pups (see above).
Statistics:
See below
Reproductive indices:
See at results
Offspring viability indices:
See at results

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed

Details on results (P0)

See for general toxicity, the repeated dose study (28-day).

No differences were detected in the percentage of breeding pairs that mated across the groups (100% in all groups). All females were detected as sperm positive during the mating period, with the exception of animal #575 in the 1 mg/kg group for which no evidence of mating was detected due to
misdiagnosis of a vaginal smear; this animal was pregnant, underwent successful parturition and was therefore considered to be sperm positive. The
number of dams that successfully underwent parturition was 9-10 in each group. One female (#558) in the vehicle control group was detected as sperm positive, but did not show any evidence of parturition or pregnancy. Another female (#581) in the 10 mg/kg group was pregnant (as detected by
implantation sites in the uterus), but never showed signs/evidence of parturition. The average number of days to achieve sperm positive status ranged from 3.5-4.1 days across treatment groups. The length of gestation was similar across all groups and averaged approximately 22 days. No significant differences in the mean number of corpora lutea/dam or total implantation sites/dam were observed.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
>= 45 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects on reproduction observed

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)

Evaluations of litter survival and offspring body weights were limited to those dams that underwent successful parturition. No statistically significant differences in mean litter size were detected across groups. The number of dead fetuses that were observed (ranged from 2-5/group) and the percentage of pups surviving to lactation day 4 were comparable across groups. In addition, no significant differences in pre-implantation, pre-natal or post-natal loss of offspring were observed across groups. Mean sex ratios (males/females) for days 0 and 4 were also unaffected by treatment. No significant differences in offspring body weights were observed. Overall mating, pregnancy, offspring survival and pup body weights were not notably affected by treatment with NTAN.

Gross external observations, following parturition on lactation day 0, were low in incidence and were noted in 4-6 fetuses (of the F1 generation) in each treatment group, including the vehicle control group. One control fetus was noted with a blunted tail on lactation days 0 and 4. All other gross external morphology observations were common observations likely associated with delivery and consisted mainly of bruising, pale skin and a lesion/scab on the neck. Two pups in the 1 mg/kg group were noted as cannibalized on day 0; one of which was missing its left hindlimb and tail (most likely due to cannibalization). None of the gross external observations were attributed to NTAN treatment.
Visceral examinations were performed on dead pups, when feasible. No visible abnormalities were noted during the visceral examinations. The stomach of one pup from dam# 590 in the 45 mg/kg group was noted to be devoid of milk; however, this observation was not attributed to NTAN treatment.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Oral administration of NTAN to Sprague-Dawley rats at doses of 1, 10 and 45 mg/kg/day resulted in overt signs of parental toxicity at the 45 mg/kg/day dose including death, clinical signs of toxicity (including convulsions, labored breathing, etc.), decreased food consumption, body weight, body weight gain, and motor activity changes (see also the 28-day repeated dose study). Overall mating, pregnancy, offspring survival and pup body weights were not notably affected by treatment with NTAN. Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity of NTAN was 10 mg/kg/day and the NOAEL for reproductive/developmental toxicity was at least 45 mg/kg/day.
Executive summary:

Nitrilotriacetonitrile (NTAN) was administered to male and female Sprague-Dawley rats by oral gavage at concentrations of 0.5, 5.0 or 22.5 mg/ml equivalent to doses of 1, 10 or 45 mg/kg/day, when administered at a constant dosing volume of 2 ml/kg. The vehicle control group received vehicle (corn oil) only. Ten rats/sex/group were selected for cohabitation (i.e., core/parental animals) and 5 additional rats/sex in the control and high dose groups were designated as recovery animals in order to evaluate reversibility of any NTAN-induced effects. The parental rats were administered NTAN for 2 weeks prior to mating, during the mating period, and until sacrifice (males: 31 doses; females: a minimum of 48 doses). Recovery animals were treated for the same duration as the parental rats, but were not mated, and were held for 14 days after treatment to evaluate recovery from any adverse treatment-related effects. Due to the spontaneous deaths of 2 high dose recovery females, only 3 unmated recovery females remained. In order to increase the size of the female high dose recovery group, one core parental high dose dam was held without treatment

for approximately 14 days and was necropsied with the recovery animals. By doing this, there was one additional high dose female (n = 4 instead of n = 3) to evaluate for recovery potential from treatment-related effects. Body weights, body weight gain and food consumption were measured during the study. In addition, clinical observations, a functional observational battery with automated motor activity, organ weights, and clinical and

diagnostic pathology were evaluated.

Four rats in the 45 mg/kg/day group died spontaneously during the study; male #536 was found dead on study day 19 (during treatment/ cohabitation), female #592 was found dead on day 3 (during treatment/pre-cohabitation), female #599 (designated as a recovery animal) was

found dead on day 30 (during the treatment period), and female #586 was found dead on lactation day 3. All four deaths were considered to be treatment-related, although microscopically the cause of death for these animals was undetermined. Treatment-related clinical signs of toxicity were limited to animals in the 45 mg/kg group (6/15 females) and were often noted within a few hours following dose administration (see also the 28 -day repeated toxicity study). Treatment-related decreases in mean food consumption were observed for male and female rats in the 45 mg/kg group during the study. These reductions in food consumption began over study days 0-4 for both sexes and continued for the remainder of the treatment period for females. Corollary reductions in body weight were also observed: mean body weight and/or body weight gain were reduced compared to concurrent control values in both sexes in the 45 mg/kg group beginning over study days 1-4 and these changes generally persisted for males and females throughout the treatment phase of the study. No treatment-related changes in clinical chemistry or hematology profiles were observed in animals treated with NTAN (see also the 28 -day repeated toxicity study).

No differences were detected in the percentage of breeding pairs that mated across the groups (100% in all groups). The number of dams that successfully underwent parturition was 9-10 in each group. No gross external or visceral observations were attributed to NTAN treatment. Overall mating, pregnancy, offspring survival and pup body weights were not notably affected by treatment with NTAN.

Absolute and relative organ weights were not considered to be affected by treatment with NTAN. Treatment-related gross necropsy findings were low/singular in incidence, limited to the 45 mg/kg group and consisted of red fluid/blood in the cranial cavity/cranium (observed in four animals that died spontaneously during the study), pigmentation of the brain, thymus and liver, urinary bladder dilatation and an enlarged thymus. No treatment-related histopathological changes were observed in the tissues examined from rats in the 45 mg/kg group; all findings were considered spontaneous or incidental changes commonly observed in rats of this age and strain.

Based on the results of this study, the no-observed-adverse-effect level (NOAEL) for parental systemic toxicity of NTAN was 10 mg/kg/day and the NOAEL for reproductive/developmental toxicity was 45 mg/kg/day.

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