Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Reproductive toxicity: category 2: This classification is correct.

Hazard statement H361: Suspected of causing delayed growth of the unborn child.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive 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
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Justification for study design:
This study is designed to provide data about the possible effect of Succinonitrile on the integrity and performance of the male and female reproductive systems in SD rats and the development of pups after exposure by drinking water. The method of this test was designed to be compatible with OECD Guideline for the Testing of Chemicals, “Two-Generation Reproduction Toxicity Study” (No.4l6, adopted in 2001).
Test material information:
Composition 1
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 SN0620140520 /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
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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 arrival. 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 performed 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.
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) .
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 birth and rearing of their pups. The mating period was continual for two weeks. In the second week of the mating period, female without successful copulation was replaced with another male animal from the 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.
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 concentrations and drinking water as untreated control was sampled. After the treatment, each concentration 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 report (Annex 3). The results indicate that the concentrations of the prepared drinking water formulation were within acceptable limits.
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) .
Details on study schedule:
shedule see attached document under background information
Dose / conc.:
100 ppm
Dose / conc.:
250 ppm
Dose / conc.:
625 ppm
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
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 same 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.
Parental animals: Observations and 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 females 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 consumption 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.
Estrous cyclicity (parental animals):
Estrus cycle evaluation
For F0 Generation and selected F1 Generation females, vaginal smears to evaluate the estrus cycle were conducted for two weeks prior to mating. For each female, vaginal smear was checked twice daily in the morning and afternoon respectively, and the check result was recorded as estrus (E) and non-estrus (NE). For estrus, most of cells in vaginal smear are epithelia with some nucleate cells observed by microscope, and for non-estrus, fewer of epithelia can be seen in vaginal smear. As estrus was found, the female was suspended the check for one time.
Sperm parameters (parental animals):
At schedule necropsy of the parental males of F0 Generation, a subset of ten males in the control group and high-dose group, and all males that had no clear evidence of mating in each group were selected for sperm analysis. At schedule necropsy of the parental males of F1 generation, a subset of ten males in the control group and all dose groups, and all males that had no clear evidence of mating in each group were selected for sperm analysis.
Litter observations:
For all pups of F0 and F1 generations, a cage-side observation of the litters was conducted only once daily except for PND 0, 4, 7, l4 and 21. On these days, pups were observed individually, and the number of live pups was recorded. On PNDO, the number of each sex, stillbirth and grossly malformed pups were recorded and evaluated.
On PNDO and 4, litter weights were determined. On PND4 after culling, and on PND 7, 14 and 21, the pups were weighed individually.
Postmortem examinations (parental animals):
For F0 and F1 generation, males were sacrificed after the end of the mating period, and females were sacrificed after all litters were weaned. All surviving parent male and female rats were euthanized by C02 inhalation followed by exsanguinations from abdominal aorta and subjected to a full necropsy and
general observation. A necropsy was performed on animal that was dead intercurrently on the death day. The necropsy included carefiilly examinations of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities and their contents of all animals, and the location, size, hardness and the color of the abnormal findings were recorded. Special attention was paid to the organs of the reproductive system. For the parous females, the number of implantation sites in the uteri and the number of corpora lutea were recorded.
Postmortem examinations (offspring):
As all litters were weaned, one male and one female pup of each litter as possibly, and all pups with external abnormalities or clinical signs were euthanized by C02 inhalation followed by exsanguinations from abdominal aorta and subjected to a lull necropsy and general observation. Gross necropsy was performed on dead pups during lactation at the time of the pups‘ death for possible defects. In the necropsy, carefully examinations of the external features of the carcass, external body orifices, the abdominal, thoracic, and cranial cavities and their contents of all animals were included, and the special attention was paid to the organs of the reproductive system. As being sacrificed, the pups were coded following the parental animals.
Statistics:
The resulting data were analyzed using the statistic methods mentioned in the responding data tables. P < 0.05 was considered as a level of significance. a) The data of clinical signs were analyzed by X2-test; b) Data of the mean body weight and mean body weight gain, water consumption in premating period were statistic analyzed by decision tree in General Toxicology Test Administration System 1.0; the data in the other period were statistic analyzed by ANOVA followed by Dunnett's test as p<0.05 or by Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-test as p<0.05;
0) Data of the absolute and relative organ weight were statistic analyzed by one factor analysis of variance (ANOVA) followed by Dunnett's test or by
Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-test as p<0.05;
d) Estrus cyclicity were evaluated by X2-test for number of acyclic animals and number of animals with prolonged estrus period, by Mann-Whitney U-test for grade analysis of length of the longest cycles, by one factor analysis of variance (ANOVA) followed by Dunnett's test or Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-test for mean cycle length and mean of the longest cycles;
e) The time of mating and the gestation length were evaluated by Mann-Whitney U-test for grade analysis, and by one factor analysis of variance (ANOVA)
followed by Dunnett's test or Kruskal—Wallis non-parametric ANOVA followed by Mann-Whitney U-test for mean time; f) The reproductive parameters were evaluated by Xz-test; g) Sperm parameters were evaluated by ANOVA followed by Dunnett's test or Kruskal-Wallis non-parametric ANOVA followed by Mann-Whitney U-test for sperm count and motility parameters; h) Incidences of pathological findings were analyzed by the Fisher’s exact probability test.
All data analysis had been done with test software in this facility or SPSS 22.0.
Reproductive indices:
Female mating index = (number of mated females/number of females placed
with males) x 100
Male mating index = (number of mated males/number of males placed with
females) X 100
Male fertility index = (number of males siring a litter/number of males placed
with females) X100
Female fertility index = (number of pregnant females/number of females
placed with males) X100
Female fecundity index = (number of pregnant females/number of mated
females) X100
Gestation index = (number of females with live pups / number of pregnant
females) X100
Offspring viability indices:
Live birth index = (number of pups born alive / number of pups born) X100
Viability index of lactation = (number of surviving pups on PND21 / number
of pups afier culling on PND4) X100
Sex ratio = (number of live male pups on PNDO/ number of live pups on
PNDO ) x 100
Pre-implantation loss = (number of corpora lutea - number of implantation
sites) X100/ number of corpora lutea
Post-implantation loss = (number of implantation sites - number of pups
livebom) X 100/ number of corpora lutea
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
no effects observed
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:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Reproductive function: estrous cycle:
no effects observed
Description (incidence and severity):
The number of females with different length of longest estrus cycle had a statistically significant difference in the mid-dose group compared with the control group (p<0.01), but the length of longest estrus cycle in most females was within 4.0 ~9 days, that is the normal cycle length for SD rats, and no difference was observed on the other estrus cyclicity parameters in the mid-dose group, so that result was considered to be without toxicological significance. No difference was observed on all estrus cyclicity parameters in the high— and low- dose groups
compared with the control group (p>0.05).
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
A statistically significantly increase on motility of sperms and ratio of rapid sperm was observed in high-dose group compared with the control group (p<0.05 or p<0.01). These results showed that the epididymal sperm of the examined males
in high-dose group was more active than the control group, and that was not
considered to be an adverse effect of the treatment of the test item. A statistically
significantly increase on motility of spenns and ratio of rapid sperm was observed
in the examined males of low-dose group compared with the control group
(p<0.05 or p<0.01), and a statistically significantly increase on area of sperm motility was observed in the examined males of low- and mid- dose groups compared with the control group (p<0.01), so it was considered that the males
which failed to become siring in low— and mid- dose groups had not gotten an
adverse effect on epididymal sperm motility.
A statistically significantly increase on the total count of epididymal sperm was
observed in high-dose group compared with the control group (p<0.01), but that
was not considered to be an adverse effect of the treatment of the test item. A
statistically significantly increase on the total count of epididymal sperm was
observed in the examined males of low-dose group compared with the control
group (p<0.01), and no statistically significant difference was observed in the
examined males of mid-dose group compared with the control group (p>0.05), so
it was considered that the males which failed to become siring in low- and mid-
dose groups had not gotten an adverse effect on epididymal sperm count.
Reproductive performance:
no effects observed
Description (incidence and severity):
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).
Key result
Dose descriptor:
NOAEL
Effect level:
625 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effect seen
Key result
Dose descriptor:
NOAEL
Effect level:
56.7 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse effect seen
Key result
Dose descriptor:
NOAEL
Effect level:
76 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse effect seen
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Several animals were observed with dehairing and scar on different position of body, but no treatment-relation was observed in all dose groups.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (No. 2611) of the mid-dose group was found dead on GD22, at necropsy, thirteen embryos were found in its uterus, and no apparent cause of death was found, so it was considered that the animal was dead because of dystocia.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 - generation (shown in Tables and Table6, Fig2)
For males, the mean body weight as grouping and in each test week had a statistically significant decrease in high-dose group compared with the control group (p<0.05 or p<0.01), at the same time, the body weight gain in the second and third weeks and the total body weight gain during the whole premating period had a statistically significant decrease in high-dose group compared with the control group (p<0.05 or p<0.01); the mean body weight in the 7m week had a statistically significantly increase in mid-dose group compared with the control group (p<0.05), at the same time, the body weight gain in the 5‘h week and the total body weight gain during the whole premating period had a statistically significantly increase in mid-dose group compared with the control group (p<0.05), that was considered to be an incidental result without toxicological significance; no significantly difference was observed on mean body weight and body weight gain in low-dose group compared with the control group (p>0.05). For females, the mean body weight fi'om grouping to the second week, and the body weight gain in the second and third weeks had a statistically significant decrease in high-dose group compared with the control group (p<0.05 or p<0.01), Q but the mean body weight and the body weight gain in the other weeks and the total body weight gain during the whole premating period had no significantly difference compared with the control group (p>0.05), so this result was considered to be because of the statistically significant decrease in the mean body weight as animal grouping in high-dose group compared with the control group (p<0.01); the body weight gain in the 3rd and 9h weeks in mid-dose group had a statistically significant difference compared with the control group (p<0.01), but no significantly difference was observed on mean body weight and the total body weight gain, so that was considered to be an incidental result without toxicological significance; the body weight gain in the 3rel week in low-dose group had a statistically significant increase compared with the control group (p<0.05), but that was considered without toxicological significance.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
For males, the mean food consumption in each test week had a statistically significant decrease in high-dose group compared with the control group (p<0.01), at the same time, the total food consumption in the premating period had a statistically significant decrease in high-dose group compared with the control group (p<0.01); the incidental increases or decreases of mean food consumption were observed in low- and mid- dose groups(p<0.05), but no statistically significant difference on the total food consumption was observed in these two groups (p>0.05), so the results were considered to be incidental without toxicological significance. For females, the mean food consumption in the 1ste, 2nd, 7lh and 10th test weeks had a statistically significant decrease in high-dose group compared with the control group (p<0.05 or p<0.01), at the same time, the total food consumption in the premating period had a statistically significant decrease in high-dose group compared with the control group (p<0.01); the incidental increases or decreases of mean food consumption were observed in low- and mid- dose groups(p<0.05 or p<0.01), but the total food consumption had no statistically significant difference compared with the control group (p>0.05), so that was considered to be an incidental result without toxicological significance
Food efficiency:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation (shown in Table 22)
For males, the mean food efficiency during the whole premating period had no
statistically significant change in all dose groups compared with the control group
(p>0.05).
For females, the incidental increases of mean food efficiency were observed in all
dose groups in the 2th and 3‘“ week (p<0.05 or p<0.01), but the average food
efficiency in the whole permating period had no statistically significant difference
compared with the control group (p>0.05), these results were considered to be
incidental without toxicological significance.
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation (shown in Table24)
For males, the mean water consumption in the 15‘, 2nd, 3rd and 5‘" test weeks had a
statistically significant decrease in high-dose group compared with the control
group (p<0.05 or p<0.01), at the same time, the total water consumption in the
premating period had a statistically significant decrease in high-dose group
compared with the control group (p<0.05); the incidental increases of mean water
consumption were observed in low— and mid- dose groups(p<0.05 or p<0.01), and
the total water consumption had a statistically significantly increase in mid-dose
group compared with the control group (p<0.05), but these results were
considered to be incidental without toxicological significance.
For females, the mean water consumption fi'om the 1St test week to the 8lh test
week had a statistically significant decrease in high-dose group compared with the
control group (p<0.05 or p<0.01), at the same time, the total water consumption in
the premating period had a statistically significant decrease in high-dose group
compared with the control group (p<0.01); the incidental increase or decreases of
mean water consumption were observed in low- and mid- dose groups, but no
statistically significant difference was observed in the total water consumption of
these two dose groups (p>0.05), so these results were considered to be incidental
without toxicological significance.
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
Description (incidence and severity):
F1 generation (shown in Table58 and Table59)
The following statistically significant difference compared with the control group
was observed on organ weight:
The absolute testes weight in males of high-dose group was statistically
significantly decreased (p<0.01), but the relative weight was comparable with the
control group because of a statistically significant decrease in the terminal body
weight.
Both of the absolute and relative ovaries weights in females of low- and mid- dose
groups were statistically significantly increased (p<0.05 or p<0.01), but no
statistically significant difference was observed in high-dose groups, this was
considered to be of no toxicological relevance.
The absolute spleen weight in males of mid— and high- dose groups was
statistically significantly increased (p<0.05), at the same time, the relative Spleen
weight in males of high~dose group was also statistically significantly increased
(p<0.01). Both of the absolute and relative spleen weight in females of all dose
groups was comparable with the control group.
The absolute kidneys weight in males of all dose groups tended to be increased,
and there were statistical significance in mid—dose group (p<0.01), at the same
time, the relative kidneys weight in males of all dose groups was also statistically
significantly increased (p<0.05 or p<0.01). Both of the absolute and relative spleen weight in females of all dose groups was comparable with the control
group.
The absolute brain weight in males and females of all dose groups was
comparable with the control group, but the relative brain weight in males of
high-dose group and in females of low-dose group was statistically significantly
increased (p<0.01) because of a statistically significant decrease in the terminal
body weight.
Both of the absolute and relative pituitary weight in females of low— and high-
dose groups were statistically significantly decreased (p<0.01). Both of the
absolute and relative pituitary weights in males of all dose groups were
comparable with the control group.
The absolute thyroid weight in females of high~dose group was statistically
significantly decreased (p<0.05), but their relative thyroid weight was comparable
with the control group, this was considered to be of no toxicological relevance.
Both of the absolute and relative thyroid weights in males of all dose groups were
comparable with the control group.
The absolute adrenals weight in males of high-dose group was comparable with
the control group, but their relative adrenals weight was statistically significantly
increased (p<0.01). Both of the absolute and relative adrenals weights in females
of all dose groups were comparable with the control group.
The organ weights of the other organs in males and females of all dose groups
were comparable with the control group.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Pup clinical observations
F0 Generation (shown in Table45) During the lactation period, many of live pups in high-dose group were observed with hypothermia, skin pale during PNDO-7, and they were recovered except for fourteen pups, those were not recovered until PND8. In addition, eight pups had emaciation, and five pups had hair late during PND8~21 in high-dose group. The incidence of all these signs had a statistically significantly increase compared with the control group (p<0.05 or p<0.01). No pup with any abnormality and sign was observed in the control group, low- and mid- dose groups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
13.1.2.4 Litter Data
F0 Generation (shown in Table37)
Twenty-three, 27, 24 and 23 females delivered a litter with at least one liveborn
pups, and two, 4, 6 and 3 females delivered a litter with stillbom pup in the
control, Iow—, mid— and high- dose groups, respectively. One female in the
mid-dose group (No. 2200) delivered with all stillbom. One female in the control
group (No. 2017) and two females in the low-dose group (No. 2105 and 2112) had complete litter loss during the lactation period. All live pups of one female in the
control group (No. 2008) and one female in the low-dose group (No. 2101) were
killed by C02 euthanasia because the dams were dead.
No statistically significant difference was observed on the mean number of
corpora lutea and implantation sites in all dose groups compared with the control
group (p>0.05). No difference was observed on the mean number of pups
delivered and liveborn in all dose groups (p>0.05). The pre-implantation loss in
high-dose group had a statistically significant decrease (p<0.05), this was
considered to be without toxicological significance; no statistically significant
effect on the pie-implantation loss was observed in low- and mid- dose groups
(p>0.05). The post-implantation loss in low-dose group had a statistically
significantly increase (p<0.01), but no statistically significant increase was
observed in mid- and high- dose group (p>0.05), so which was considered as an
incidental result based on the absence of dose relation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Pup body weights
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).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
13.1.2.6 Sexual Maturation (shown in Table49)
For the males of F1 generation, no statistically significant difference was observed on the day of achievement of preputial separation and the mean body weight on this day in all dose groups compared with the control group (p>0.05). For the females of F1 generation, no statistically significant difference was observed on the day of achievement of vaginal opening and the mean body weight on this day in all dose groups compared with the control group (p>0.05).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
13.1.3.4 Organ Weight ofPups
The following statistically significant difference compared with the control group was observed on organ weight of pups: F0 Generation (shown in Table66 and Table67) The absolute spleen weight in male and female pups of high-dose group was comparable with the control group, but their relative spleen weight was
statistically significantly increased (p<0.01). The absolute brain weight in male and female pups of high-dose group was statistically significantly decreased (p<0.05), but their relative brain weight was comparable with the control group, this was considered to be of no toxicological relevance.
Gross pathological findings:
no effects observed
Description (incidence and severity):
13.1.3.5 Macroscopic Observations of Pups Stillbom and dead pups (shown in Table70) For F0 and F1 generation, no abnormalities were observed in stillborn and dead pups. Scheduled necropsies (shown in Table7l and Table72) For F0 and F1 generation, no abnormalities were observed in pups selected for necropsy of all dose groups.
Histopathological findings:
not examined
Description (incidence and severity):
No microscopic observation was conducted for no gross lesion observed in the selected pups.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
F1 Generation (shown in Table46)
During the lactation period, in high-dose group, most of live pups were observed
with skin pale during PNDO-7, and they were recovered except for thirty-two pups,
those were not recovered until PND9; part of pups had emaciation during PNDO-l4, and they were recovered except for three pups; seven pups had
dehairing on several positions during PND14-16; four pups had sparse fur from
PND14—15, and all had not recovered as being weaned; one pup had dehairing on
neck during PND21-28; the incidence of the signs including skin pale, emaciation,
deharing on several positions and Sparse fur had a statistically significantly
increase compared with the control group (p<0.05 or p<0.01). In mid-dose group,
eight pups had dehairing on back from PND14, and seven had recovered on
PND16, the last one had recovered on PND18, the incidence had a statistically
significantly increase compared with the control group (p<0.01), but that was
considered as an incidental result because that the ratio of pups with clinical
symptom had no significantly increase; three pups had skin pale during PND4-5,
the incidence had no statistically significant difference compared with the control
group (p>0.05). Eight pups in the control group had alopecia during PND14-17,
and they became to be sparse fur on head from PNDIS to be scheduled killed. No
pup with any abnormality and sign was observed in low~dose group.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
F1 Generation (shown in Table40)
There were six, 3, 17 and 5 pups stillborn in the control group and low-, 1nid-,
high- dose groups, respectively. The live birth index in mid-dose group had a
statistically significant decrease (p<0.05), but no statistically significant difference
was observed in low— and high- dose group (p>0.05), so which was considered as
an incidental result based on the absence of dose relation. Zero, 1, 2 and 8 pups
respectively in the control, low-, mid- and high- dose groups were dead in the
lactation period. The viability index of lactation in high-dose group had a
statistically significant decrease (p<0.01). No statistically significant effect on the
viability index of lactation was observed in low- and mid- dose groups (p>0.05).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
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).
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
effects observed, non-treatment-related
Description (incidence and severity):
13.1.2.6 Sexual Maturation (shown in Table49)
For the males of F1 generation, no statistically significant difference was observed on the day of achievement of preputial separation and the mean body weight on
this day in all dose groups compared with the control group (p>0.05).
For the females of F1 generation, no statistically significant difference was
observed on the day of achievement of vaginal opening and the mean body weight
on this day in all dose groups compared with the control group (p>0.05).
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
F1 generation (shown in Table68 and Table69)
The absolute spleen weight in male pups of mid-dose group was statistically
significantly decreased (p<0.05), but their relative spleen weight was comparable
with the control group, this was considered to be of no toxicological relevance.
The absolute thymus weight in female pups of high-dose group was statistically
significantly decreased (p<0.05), but their relative thymus weight was comparable with the control group, this was considered to be of no toxicological relevance.
The absolute brain weight in male and female pups of all dose groups was
comparable with the control group, but the relative brain weight in female pups of
mid-dose group was statistically significantly increased (p<0.05), this was
considered to be of no toxicological relevance without dose relation.
Gross pathological findings:
no effects observed
Description (incidence and severity):
13.1.3.5 Macroscopic Observations of Pups
Stillbom and dead pups (shown in Table70)
For F0 and F1 generation, no abnormalities were observed in stillborn and dead pups.
Scheduled necropsies (shown in Table7l and Table72)
For F0 and F1 generation, no abnormalities were observed in pups selected for necropsy of all dose groups.
Histopathological findings:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Reproductive effects observed:
no

Classification

The pups in the high dose group of the 2 generation reproductive toxicity study did not thrive as well as the pups in the other dose groups and the control in the first postnatal days and suffered from a temporary growth delay. This was also reflected in a lower body weight of the pups of the F1 generation and at the start of the premating period of the F1 generation.

The lowest observed adverse effect (LOAEL) is delayed growth in the offspring at a maximum drinking water level of 625 ppm (males 56.7-72.9 mg/kg/day and females 76.0 -85.9 mg/kg/day).

Effects on sexual function and fertility, and malformations and mortalities in the offspring were not observed at this dose level. The no observed adverse effect level was found to be 250 ppm in drinking water (26-29 mg/kg/day in males and 33-35 mg/kg/day in females) in the reproductive toxicity study.

In a prenatal developmental study malformations in the offspring were even not observed at a drinking water level of even 195 mg/kgbw/day (1200 ppm in drinking water). At these dose level maternal toxicity was observed.

Conclusions:
Based on the results above, it is concluded that, 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 56.7 +/- 5.0 mg/kg/day in F0 generation, and 729 +/- 9.4 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 76.0 +/- 8.8mg/kg/day in F0 generation, and 85.9 +/- 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.
Executive summary:

Introduction. This study is designed to provide data about the possible effect of Succinonitrile on the integrity and performance of the male and female reproductive systems in SD rats and the development of pups after exposure by

drinking water. The method of this test was designed to be compatible with OECD Guideline for the Testing of Chemicals, “Two-Generation Reproduction Toxicity Study” (No.4l6, adopted in 2001). Method. This study was conducted in SD rats and all animals were SPF grade. In this study, twenty-eight rats/sex/group were exposed by drinking water to 0, 100,

250 and 625ppm Succinonitrile respectively. In F0 generation, males and females were exposed to the test item for ten weeks prior to mating, two weeks of the mating period; after that, males were killed for sperm analysis and necropsy, and females were exposed continuously during the gestation and lactation period until all pups were weaned. 28 rats/sex/group were selected from these pups as F1 generation. In F1 generation, males and females were exposed to the test item at the same doses with their parents, including the premating period of at least 10 weeks, mating period of at most 2 weeks, gestation and lactation period (for females), the males were killed for sperm analysis and necropsy after mating period, and the females were killed for necropsy after all pups were weaned. In two generations, one male and one female pup of each litter as possibly were selected for necropsy.

During the test, the clinical observations were made, the body weight, water consumption, and food consumption were weighed; the estrus cycle in F0 and F1 Generation females was evaluated prior to mating; the growth and developmental

parameters of pups were evaluated, and the sexual maturation was observed in the pups of F0 generation. All parental animals, part of live pups and dead pups were macroscopically examined for any abnormalities and pathological changes. The histopathology examination was made for the reproductive organs of the parental animals in the control and high-dose groups, and all gross lesions.

Results. The water consumption in animals of the high-dose group was influenced based on the results that the water consumption in males of F0 Generation and both sexes of F1 Generation had a statistically significant decrease during the

pre-mating period. But it was considered because of poor taste of the drinking water incorporated with the test item.

No treatment—relation clinical observation was found in this test.

The mean body weight in males of F1 Generation in high-dose group had a continuously statistically significant decrease during the pre-mating and mating periods. No treatment-related effects were observed on the mean body weight of

the other parental animals in the test. At the dose of 625ppm, the food consumption had a significant decrease in males of two generations during the premating period and females of F1 Generation during the premating and gestation periods. No treatment-related effects were observed on the food efficiency of parental animals in premating period.

For the parental animals, estrus cyclicity and sperm parameters were not adversely effected by exposure to the test item in two generations. For males in two generations, the mating index and fertility index had no statistically significant difference in all dose groups compared with the control group. For females in two generations, the mating index, fertility index and

fecundity index had no statistically significant difference in all dose groups compared with the control group, at the same time, no difference was observed on precoital interval, gestation index, gestation length in all dose groups; in two

generations, no treatment-related effect was observed on the number of corpora lutea, number of implantation sites, number of pups delivered and liveborn, the pre—implantation loss and post-implantation loss.

Both the viability index of lactation in the low-dose group of F0 generation and the live birth index in the mid-dose group of F1 generation had a statistically significant decrease, but no dose relation on these effects was observed in two

generations, at the same time, no other significant effect about pups including body weight and clinical sign was observed in the low— and mid- dose groups, so it is considered that these results are not a toxic effect. For the pups of two generations in the high-dose group, the number of pups with signs, including hypothermia, skin pale, emaciation and deharing, etc, had a statistically significant increase and most of these signs were recovered in the first week of the lactation

period, the mean body weights of live pups in part time of lactation had a significantly decrease, and the viability index of lactation in F1 generation had also a statistically significant decrease, but that in F0 generation is not significantly decreased, so based on these results, it is considered the test item can lead to the delayed growth of pups at the dose of 625ppm, but can‘t directly lead to the death of pups.

No treatment-related effect was observed on the absolute and relative organ weights of all parental animals and pups selected for necropsy. At the necropsy of all parental animals and pups, no treatment-related gross change was observed; in microscopic examination, no toxicologically relevant change was observed in the reproductive organs of the examined parental animals. Conclusion. According to the method in OECD Guideline for the Testing of Chemicals, “Two-Generation Reproduction Toxicity Study” (TG416, adopted in 2001), this study was conducted in SD rats. 28 rats/sex/group were exposed continuously by drinking water to 0, 100, 250 and 625ppm Succinonitrile

respectively for ten weeks prior to mating, two weeks of the mating period, the gestation period and lactation period until all pups were weaned. Afier that, 28 rats/sex/group were selected fiom pups of F0 Generation and exposed to the test

item by drinking water at the same closes with their mothers following the same schedule.

Based on the results above, it is concluded that, 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 56.7 +/- 5.0 mg/kg/day in F0 generation, and 729 +/- 9.4 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 76.0 +/- 8.8mg/kg/day in F0 generation, and 85.9 +/- 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.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LOAEL
Study duration:
chronic
Species:
rat

Effects on developmental toxicity

Additional information

Pregnant hamsters were treated at GD8 with a single intraperitoneal injection of succinonitrile. After sacrifice on GD 11, it was determined that the incidence of resorptions was slightly affected at 365 and 500 mg/kg bw, but not significantly. High incidence of malformations in offspring (between 60-80% at 365 and 500 mg/kg bw was noted. Most frequent malformations were neural tube defects (89% of total malformations), including exencephaly and encephalocoele. The crown-rump lengths of offspring of dams treated with 182 mg/kg or more were smaller than controls in a dose-related matter (statistically significant at 242, 365 and 500 mg/kg bw).

Therefore, it was concluded that a single intraperitoneal injection on GD8 in hamster was teratogenic with a NOAEL of 121 mg/kg bw. The NOAEL for maternal toxicity was 242 mg/kg bw based on clinical signs observed.

Justification for classification or non-classification

Reproductive toxicity: category 2: This classification is correct.

Hazard statement H361: Suspected of causing delayed growth of the unborn child.