Succinonitrile

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 416

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Succinonitrile

EC Number:

203-783-9

EC Name:

Succinonitrile

Cas Number:

110-61-2

Molecular formula:

C4H4N2

IUPAC Name:

butanedinitrile

Details on test material:

- Analytical purity: 97%
- Source: Aldrich Chemical Company, Milwaukee, WI, USA

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DSM Engineering Plastics B.V. lotnumber SN0620140
520 /SN0620140722
- Expiration date of the lot/batch: resp. 2016.07.01, 20170722
- Purity test date: resp. 99.93%, 99.89%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in a cool area (IO-20°C). Containers that have been
opened must be filled with dry nitrogen for at least 1min and then sealed. Be careful not to import any
water or impurities during the filling and sealing course.
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: Easily soluble in cold water

FORM AS APPLIED IN THE TEST white waxy solid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Strain: SPRAGUE DAWLEY (SD)
Grade: SPF
Supplier: Beijing Vital River Laboratory Animal Technology Co., Ltd.
Test Animals Quality Certification of supplier: SCXK (Jing) 2012—0001
Certificate number of animals: 11400700149820
Justification: Rat is the preferred species for two-generation reproduction toxicity
study and is accredited in the Guideline.
Number of animals: Two hundred and sixty rats with 130 females and 130 males
were purchased, and 224 rats with 112 females and 112 males were selected in the test.
Animal age on arrival: 35-41 days old.
Body weight range: 114.04-155.51g for females and 133.17-183.33g for males on arrival.
Physical check-up and acclimatization: All animals were checked for health within 24 hours after arri
val. The animals meeting the quality requisition were
acclimated to the laboratory conditions for nine days prior to the experimental start date. All animals
were weighed and identified with the special animal
markers on fin, and were signed on the cage cards at the same time. Clinical observations were per
formed daily till grouping. All animals were weighed again on the final day of the acclimatization, the
body weight range was 1 19.66-206.9l g for females and 20704-29201 g for males.

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

The oral route (via the drinking water) was selected for administration in this study. For F0 Generation
animals, the administration was continued during the
premating period of near 10 weeks, mating period of 2 weeks, the gestation and lactation period (for
females) until the termination. For Fl Generation animals
selected for mating, the administration was continued from weaning to termination at the same doses
with their parents, including the premating period of
10 weeks, mating period of 2 weeks, the gestation and lactation period (for females) .

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

In this study, the concentration and stability of each drinking water formulation was checked for four
times, including the first, the last preparation and twice preparations for once per near three months
in the course of the test. As being checked, the prepared drinking water formulation of different conc
entrations and drinking water as untreated control was sampled. After the treatment, each concentra
tion was analyzed for three times and the untreated control was analyzed
twice. To be considered acceptable, the actual results for the analysis of the dosing formulation are
to be within i 20% of the nominal concentration, and the actual results on the fourth day after the
preparation are to be within +/- 20% of the results at the time of the preparation. The details about the methods used and the results obtained were shown in the analysis report attached in this final re
port (Annex 3). The results indicate that the concentrations of the prepared drinking water formulation
were within acceptable limits.

Details on mating procedure:

At the end of the premating period, two weeks of the mating period began for F0 Generation and
selected F 1 Generation animals. Each female was placed with one male from the same group in the
mating cage. In every morning during the mating period, vaginal plugs was checked in each mating
cage, and for females with the presence of vaginal plugs, vaginal smears was checked for sperm.
The presence of sperm was considered as the evidence of successful copulation. The day on which
sperm was detected was considered as GDO. The mated females were caged individually for the birt
h and rearing of their pups. The mating period was continual for two weeks. In the second week of t
he mating period, female without successful copulation was replaced with another male animal from t
he same group (a male that already had successfully mated with another female.) After the end of the
mating period, females without successful copulation were
caged individually until sacrifice (more than 21 days after the last day of the mating period), and
sperm positive females that turn out to be non-pregnant were
killed for necropsy at the same time.

Duration of treatment / exposure:

The oral route (via the drinking water) was selected for administration in this study. For F0 Generation
animals, the administration was continued during the
premating period of near 10 weeks, mating period of 2 weeks, the gestation and lactation period (for
females) until the termination. For Fl Generation animals
selected for mating, the administration was continued from weaning to termination at the same doses
with their parents, including the premating period of
10 weeks, mating period of 2 weeks, the gestation and lactation period (for females) .

Duration of test:

shedule see attached document under background information

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 ppm

Doses / concentrations 2

Dose / conc.:

250 ppm

Doses / concentrations 3

Dose / conc.:

625 ppm

No. of animals per sex per dose:

28

Control animals:

yes, concurrent no treatment

Details on study design:

Refer to the doses conducted in repeated dose 90-day oral toxicity study of succinonitrile in rats and
the results of its preliminary test (Study No.G1557B002A), three dose levels were used in this study
including 625ppm, 250ppm and 100ppm. A concurrent control group (Oppm) was included at the sam
e time. The test item was administered via the drinking water.
In repeated dose 90-day oral toxicity study of succinonitrile in rats, three dose levels are 750ppm,
250ppm and 100ppm. The results of its preliminary test indicated that one animal was dead on Day
17 and showed emaciation at the dose of 1000ppm administered via the drinking water. There were
no deaths or symptoms at the dose of 500ppm during the dosing period. The mean body
weights in the 1000ppm group were slightly less than those of the controls. Water consumption by
males and females in the 1000ppm was more than 10% less than that by the controls.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning hours, detailed observation once a week
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily in the morning hours, detailed observation with
handling was made once per week
BODY WEIGHT: Yes
- Time schedule for examinations: For F0 and F1 generation, all animals were weighed at grouping
, and once per week during the premating period; males were weighed once per week during the
mating period; mated females were weighed on gestation day (GD) 0, 7, 14 and 21; puerperal femal
es were weighed on postnatal day (PND) 0, 4, 7, 14 and 21
during lactation period. The animals were weighed on their scheduled necropsy
date.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
During the premating and gestation period, the ration food was added once weekly.
The added food weight was 500 +/- 10g; in the premating period, and 200 +/- 10g in the
gestation period. The food was weighed again three days (72h +/-5h) later as surplus food weight.
The results were expressed in g per animal per day.
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
During the premating period, the ration water consumption was determined once weekly at the same
time as the food consumption determined. In the gestation and lactation period, the ration water c
onsumption of the mated female animal was determined once weekly at regular time until weaning.
The added water volume was about 500mL in the premating period, and the enough volume of water
was added in the gestation and lactation period. The bottles filled with water were weighed before to
give animal, and were weighed again three days (72hi1.5h) later for surplus water weight. The results
were expressed in g per animal per day. Water bottles were refreshed at least every four days and
marked with the cards
with the information as same as the cage cards. Water bottles were inspected twice
daily at the beginning and the end of work.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Body weight and weight changes:

effects observed, non-treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Gross pathological findings:

effects observed, non-treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

no effects observed

Maternal developmental toxicity

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

effects observed, non-treatment-related

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

625 ppm

Based on:

test mat.

Basis for effect level:

other: no adverse effect seen

Results (fetuses)

Fetal body weight changes:

not examined

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): effects observed, treatment-related
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): F0 Generation (shown in Table47)
The mean body weight of live pups in high-dose group had a decrease during the
lactation period, and the decrease on PND7 was statistically significant compared
with the control group (p<0.01). The mean body weight of live pups in low— and
mid- dose groups had no statistically significant difference during the lactation
period compared with the control group (p>0.05).
F1 Generation (shown in Table48)
The mean body weight of live pups in high-dose group had a statistically
significant decrease on PND7, PND14 and PND21 compared with the control
group (p<0.01 or p<0.05). The mean body weight of live pups in low- and mid-
dose groups had no statistically significant difference during the lactation period
compared with the control group (p>0.05).

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

F0 Generation (shown in Table3 5)
Twenty-three, 27, 25 and 23 pregnant females survived delivery, and twenty-three,
27, 24 and 23 females delivered a litter with liveborn pups in the control, low-,
mid- and high- dose groups, reSpectively. One female in the control group and two
females in the high-dose group were dead during the delivery. One female in the
control group was without delivery, but with implantation sites observed at
necropsy. The gestation index had no statistically significant difference in all dose
groups compared with the control group (p>0.05).
No statistically significant difference was observed on gestation length in all dose
groups compared with the control group (p>0.05).
F1 Generation (shown in Table3 6)
Twenty-five, 28, 26 and 26 pregnant females survived delivery, and all delivered a
litter with liveborn pups in the control, low-, mid- and high» dose groups,
respectively. One female in the mid-dose group was dead during the delivery. The
gestation index had no statistically significant difference in all dose groups
compared with the control group (p>0.05).
No statistically significant difference was observed on gestation length in all dose
groups compared with the control group (p>0.05).

Changes in sex ratio:

no effects observed

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 ppm

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Applicant's summary and conclusion

Conclusions:

under the conditions of the
two-generation reproduction toxicity study in rats, the No Observed Adverse
Effect Level (NOAEL) for parental toxicity to males, for exposure to
Succinonitrile by drinking water, is considered to be 625ppm with the daily actual
chemical intake of S6.7:I:5.0 mg/kg/day in F0 generation, and 72919.4 mg/kg/day
in F1 generation; the NOAEL for parental toxicity to females is considered to be
625ppm with the daily actual chemical intake of 76.0dz8.8mg/kglday in F0
generation, and 85 .9i 8.4mg/kg/day in F1 generation to females.The LOAEL for reproductive toxic effects over 2 generations (temporary growth
retardation in pups) by exposure to succinonitn'le via drinking water, is considered
to be 625 ppm and the NOAEL is considered to be 250 ppm.