Ethylenediaminetetraacetonitrile

Toxicity to reproduction

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:

June-July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Batch number is given; CoA has been added to the report. Well performed and reported study according to guideline/standard (although guideline used has not been mentioned).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Principles of method if other than guideline:

In the report it was not indicated which guideline was used but the study essentially followed OECD guideline 421

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethylenediaminetetraacetonitrile

EC Number:

227-290-3

EC Name:

Ethylenediaminetetraacetonitrile

Cas Number:

5766-67-6

Molecular formula:

C10H12N6

IUPAC Name:

2-({2-[bis(cyanomethyl)amino]ethyl}(cyanomethyl)amino)acetonitrile

Details on test material:

Name of test compound: EDTN
Chemical name: N.N,N,N-Tetracyanomethyl-1,2-ethanediamine
Lot nos: CFC-6531 (for analytical development) and CFC-7336 (for dose formulations)
Receipt: 22 March 2005 and 29 April 2005, respectively
Appearance: white powder
Storage: room temperature (22±3 degr. C) protected from light
Composition of Lot no. CFC-7336 (see CoA added to the report):
- EDTN: > 99.8% (GC-MS)
- EDTN: 99.98% (NMR 600 MHz)
- HCN: 0.9 ppm

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, NY, USA
- Age at study initiation: (P) 8 wks
- Weight at study initiation: (P) Males: ca. 260±13 g; Females: ca. 180±10 g
- Fasting period before study: no
- Housing: individually exceot during mating (1:1)
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period:


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-26
- Humidity (%): 30-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 14 June 2005 (randomization) To: the end of July 2005 (no exact end date given)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 3% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
16.67, 83.3 and 250 mg/mL (later reduced to 166.6 mg/mL for females) in 3% tween 80


VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: see above
- Amount of vehicle (if gavage): 3 mL/kg bw
- Lot/batch no. (if required): Fisher lot# 002016
- Purity: no info

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 1 week
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Mating was completed within 1 week.
- After successful mating each pregnant female was caged: individually
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of EDTN in aqueous 3% tween 80 was measured using gas chromatography (GC). Aliquots of the formulations
were diluted and analyzed with a flame ionization detector (FID). Parameters evaluated in the method validation included GC system precision, specificity, accuracy and linearity. Following validation, the method was used to determine concentration, homogeneity
and stability of the dose formulations. Dose formulations were prepared approximately every week at graded concentrations of
16.67, 83.3 and 250 mg/ml. After 13 days, the high dose given to the females was reduced from 750 mg/kg to 500 mg/kg and so
administered to the high dose females for the remainder of the study. Samples of the dose formulations from the first two
preparations, the middle preparation and the last preparation were analyzed for concentration. Homogeneity was also determined
for samples collected from the first two preparations of the low and high dose formulations. The dose formulations proved to be
homogeneous and stable for up to 44 days at room temperature; while standard stability was determined to be at least 70 days.
The analytically-determined concentrations of the dose formulations were within the protocol specified acceptance criteria for
suspensions (i.e. 15% of target).

Duration of treatment / exposure:

males: 2 weeks prior to mating, 1 week of mating, and 2 weeks thereafter (total of 5 weeks)
females: 2 weeks prior to mating, 1 week of mating, through gestation and lactation (up to day 4; total of 6 weeks)

Frequency of treatment:

daily

Details on study schedule:

Only parent animals were dosed, the F1 generation (pups) was killed on day 4.

Doses / concentrations

Remarks:

Doses / Concentrations:

Basis:
actual ingested
50, 250, 750 mg/kg bw (after 2 weeks the high dose was lowered to 500 mg/kg for females only)

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: no info
- Rationale for animal assignment (if not random): randomization upon BW

Positive control:

Not used

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: pretest and weekly

BODY WEIGHT: Yes
- Time schedule for examinations: pretest, day -1, and weely thereafter. Pregnant females also on gestation days 0, 7, 14 and 20,
dams ans litters on postnatal day 0 and day 4. In addition, fasted BW were collected priro to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal: weekly for males (except during cohabitation), for females: weekly prior to mating, on gestation days 0, 4, 7, 14 and 20, and on postpartum days 0 and 4.

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

OTHER: no

Oestrous cyclicity (parental animals):

No

Sperm parameters (parental animals):

Parameters examined in male parents:
testis weight, epididymis weight, and histopathology on these 2 organs

Litter observations:

STANDARDISATION OF LITTERS: not done as all pups were sacrificed on day 4 postpartum

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain

GROSS EXAMINATION OF DEAD PUPS:
yes, for gross external abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals: ca. 35 days after study start
- Maternal animals: All surviving animals: day 4 postpartum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed (*)
- vagina
- ovaries with oviducts (*)
- pituitary gland
- cervix
- testes (*)
- epididymides (*)
- liver (*)
- seminal vesicles
- prostate
- coagulating gland
- uterus
- target organs
Microscopic examination was performed on ovaries, testes and epididymides (10/sex/group) of control and high dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at 4 days of age.
- These animals were subjected to postmortem macroscopic examinations.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS: not done

Statistics:

Data were manually collected, entered into Microsoft Excel®, and means and standard deviations were calculated using the same
software. Data were analyzed for homogeneity of variance using Levene's test. When the variances were homogeneous (p >
0.001), the data were further analyzed by one-way analysis of variance (ANOVA). When main effect differences were found, all
post-hoc comparisons between the test material-treated and vehicle controls were conducted using Dunnett’s test. In addition,
mating performance and absolute pup survival were analyzed using Chi-Square. Statistical significance was declared at p ≤0.05.
Data was manually collected, entered into Excel®, and means and standard deviations were calculated using the same software.
Statistical analysis was performed using Systat® 10.

Reproductive indices:

Mating = (# Successful Parturition ÷ # Selected for Littering) x 100
%Pre-implantation loss = [(Total Corpora Lutea – Total Implants) ÷ Total Corpora Lutea] x 100
%Post-implantation loss = [(Total Implants – Total Born) ÷ Total Implants] x 100

Offspring viability indices:

Number alive on Day 0, Total number of males born alive, Total number of females born alive, Ratio of males to females
Total number of pups born, Number of stillborn fetuses, % alive on Day 0
Number alive on Day 0, Number alive on Day 4, % alive on Day 4, % surviving

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:

not specified

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)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One high dose female showed signs of ataxia, lethargy, tremors, discolored fur, hunched posture, piloerection and was cold to
touch, therefore, the animal was sacrificed in a moribund condition. Other females in the high dose group showed clinical signs of
emaciation (thin), were unkempt and had discolored fur; these signs were indicative of overt toxicity as such the dose was
lowered from 750 mg/kg/day to 500 mg/kg/day for the high dose females only starting on day 13 of treatment and continuing for the
remainder of the study.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Statistically significant differences in body weight/body weight gain and food consumption were not always achieved; however, a
trend for reduced body weight/body weight gain and food intake was seen in the high dose group.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Intake per gavage.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Not examined.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No statistically significant differences in the percent mated were noted between the treated and control groups. The number of
dams that successfully underwent parturition (successful mating outcome) was 89% or greater for each group (100% in the control and low dose groups, 91% in the mid dose group and 89% in the high dose group). The average length of gestation was
unaffected by treatment and was approximately 22 days for each group.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Treatment with EDTN did not result in any significant changes in reproductive organ weights. Liver-to-body weight ratios were
significantly elevated in the low and mid dose males; this change was not considered to be biologically significant due to the small
maginitude and lack of dose response relationship.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Kidney lesions (pigmentation, mottled) were noted at a higher incidence in the high dose females; this may have been treatment-
related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects were noted in the tissues examined (i.e. ovaries, testes or epididymides).

OTHER FINDINGS (PARENTAL ANIMALS): none

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: mortality (1 female); body weight; food consumption

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

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 specified

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
Percent survival on Postnatal Day 4 was significantly reduced in the high dose group compared to controls (86 vs. 99%); this
reduction was the result of a single high dose dam which had only 3 of 14 pups survive until postnatal day 4. Fetal loss was not
observed in any other high dose dam over postnatal days 0-4; as such, this was considered an isolated incidence and may have
been a residual effect of the toxicity seen earlier in this study since this dam did not gain weight during the first 2 weeks of
treatment.

CLINICAL SIGNS (OFFSPRING)
No data.

BODY WEIGHT (OFFSPRING)
No changes.

SEXUAL MATURATION (OFFSPRING)
Not examined, pups were killed on day 4.

ORGAN WEIGHTS (OFFSPRING)
Not examined.

GROSS PATHOLOGY (OFFSPRING)
No data.

HISTOPATHOLOGY (OFFSPRING)
Not examined.

OTHER FINDINGS (OFFSPRING)
None:

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Sex:

male/female

Basis for effect level:

other: Pup survival was reduced in the high dose group on Postnatal Day 4, but this was the result of a single dam and considered an isolated event. There were no other changes.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these findings, the no-observed-adverse-effect level (NOAEL) of EDTN for reproductive toxicity is at least 500 mg/kg, while the
no-observedadverse-effect level for maternal (and paternal) toxicity was 250 mg/kg.

Executive summary:

The study was performed essentially according to OECD 421 (although this was not mentioned in the report). EDTN was formulated in 3% tween 80 (vehicle) and was administered by oral gavage at dose levels of 0 (vehicle control), 50, 250 or 750 mg/kg/day (after 2 weeks of treatment, the high dose was lowered to 500 mg/kg/day for females only) to 12 Sprague-Dawley rats/sex/group for the assessment of reproductive and developmental toxicity. Doses were administered at a constant volume of 3 ml/kg for 2 weeks prior to mating, during an approximately 1 week of mating, through gestation and lactation until sacrifice for a total of 5 weeks of dosing for males and approximately 6 weeks for females. Parental food consumption, body weights, body weight gain, reproductive performance, organ weights and histopathology were evaluated during the study, along with offspring body weights and survival.

One high dose female was sacrificed in a moribund condition; clinical signs for this female consisted of ataxia, lethargy and being cold to touch. Necropsy findings for the same female were unremarkable. No other treatment-related deaths occurred. Other females in the high dose group showed clinical signs of emaciation (thin), had unkempt appearance and discolored fur; these signs were indicative of toxicity. Based on these

clinical signs, the dose for the high dose females was lowered to 500 mg/kg/day. Administration of EDTN resulted in overt signs of parental toxicity in the high dose group as indicated by reduced food consumption, body weights, and body weight gains and one treatment-related death in the high dose females. Treatment with EDTN did not result in any reproductive organ weight changes or in any underlying histological changes in the ovaries, testes or epididymides.

No statistically significant differences were detected among the groups in mean corpora lutea, implantation sites, litter size, and mean number of total pups born or pups born alive on postnatal day 0. Percent survival on Postnatal Day 4 was significantly reduced in the high dose group compared to controls (86 vs. 99%); this reduction was the result of a single high dose dam which had only 3 of 14 pups survive until Postnatal Day 4. Fetal loss was not observed in any other high dose dam over postnatal days 0-4; as such, this was considered an isolated incident. The incidence of fetal loss in the other groups over postnatal days 0-4 was 1, 2 and 1 pup in one litter per group for the control, low and mid dose groups. No other differences in pup survival were observed in any other group. Offspring body weights and growth were similar across all groups and unaffected by treatment.

In conclusion, overt maternal toxicity was seen in the high dose dams, as evidenced by reduced maternal body weight; while litter size (Postnatal Day 0) and offspring body weights were unaffected by treatment and were comparable to controls on Postnatal Days 0 and 4. Although offspring survival was reduced in the high dose group this was confined to a single dam and considered an isolated event. Based on these findings, the no-observed-adverse-effect level (NOAEL) of EDTN for reproductive toxicity is 500 mg/kg (the highest dose tested), while the no-observed-adverse-effect level for maternal toxicity is 250 mg/kg.