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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
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
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)
Principles of method if other than guideline:
range-finding test, primarily according to OECD 407
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 oral 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.
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
Doses / concentrations
Remarks:
Doses / Concentrations:
1, 10, 45 mg/kg bw
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
Details on study design:
See below
Positive control:
Not used

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes, weekly

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: sodium pentobarbital
- Animals fasted: Yes
- How many animals: 5 rats/sex/group (main and recovery)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: 5 rats/sex/group (main and recovery)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males after 29 doses, females after post-partum Day 4
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity

OTHER: see at developmental/reproduction toxicity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
See below

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY: one male and three females were found dead. Clinical signs consisted of discoloration around the mouth and nose, discolored/wet inguinal fur, convulsions, labored breathing, irritability, head weaving, loss of righting reflex, repetitive behavior, cyanosis, salivation, hypoactivity, discolored paws and rough hair coat. However, these signs were primarily on a single but different day of the study, which is suggestive of the acute nature of this observed toxicity.

BODY WEIGHT AND WEIGHT GAIN: reduced at 45 mg/kg bw

FOOD CONSUMPTION: reduced at 45 mg/kg bw

FOOD EFFICIENCY: not measured

WATER CONSUMPTION: not measured

OPHTHALMOSCOPIC EXAMINATION: not measured

HAEMATOLOGY: no treatment-related changes

CLINICAL CHEMISTRY: no treatment-related changes

URINALYSIS: not measured

NEUROBEHAVIOUR: No treatment-related differences in grip strength or F.O.B. parameters were noted across treatment groups for core or recovery animals. Statistically significant differences in motor activity assessments were observed for males in the 45 mg/kg group, compared to controls, and included decreased totals for move episodes, stereotypy 1 moves and episodes, and stereotypy 2 moves and time, suggestive of a change in pattern of behavior for these animals.The mean values for these parameters generally did not fall within their respective concurrent control ranges and were likely treatment-related. However, this change was only present at the dose level that produced overt systemic toxicity (death). Following the recovery period, total move episodes remained significantly decreased for males of the 45 mg/kg group, compared to controls, indicating that effects on total move episodes were not reversible within the 14-day period following cessation of dosing. No significant differences in motor activity parameters were noted for females across treatment groups for core or recovery animals.

ORGAN WEIGHTS: no treatment-related changes

GROSS PATHOLOGY: limited to the 45 mg/kg group; 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. Pigmentation of the thymus was also observed in one male of the 45 mg/kg group animal following the recovery period; whereas, one female in the 45 mg/kg group was observed with this finding at the end of the treatment period.

HISTOPATHOLOGY: NON-NEOPLASTIC, no treatment-related changes

Effect levels

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: death, clinical signs, decreased food intake and BW, and motor activity changes at 45 mg/kg bw

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the no-observed- adverse-effect level (NOAEL) for parental systemic toxicity following oral administration of NTAN to Sprague-Dawley rats for 31 days (males) or at least 48 days (females) was 10 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. These clinical signs consisted of discoloration around the mouth and nose, discolored/wet inguinal fur, convulsions, labored breathing, irritability, head weaving, loss of righting reflex, repetitive behavior, cyanosis, salivation, hypoactivity, discolored paws and rough hair coat. However, these signs were primarily on a single but different day of the study, which is suggestive of the acute nature of this observed toxicity. 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.

No treatment-related differences in grip strength or F.O.B. parameters were noted across treatment groups for core or recovery animals, or in motor activity parameters for females. Statistically significant differences in motor activity assessments were observed for males in the 45 mg/kg group, compared to controls, and included decreased totals for move episodes, stereotypy 1 moves and episodes, and stereotypy 2 moves and time, suggestive of a change in pattern of behavior for these animals. Following the recovery period, total move episodes remained significantly decreased for males of the 45 mg/kg group, compared to controls. These changes in motor activity were likely treatment-related but were only present at the dose level that produced overt systemic toxicity (death).

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.

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