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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 SN0620140520
- Expiration date of the lot/batch: 2016.07.01
- Purity test date: 99.93%

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 species / strain selection:
Rats are the species of choice as historically used for the safety evaluation studies and are specified in the appropriate test guidelines.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Beijing Vital River Laboratory Animal Technology Co., Ltd.
- Females (if applicable) nulliparous and non-pregnant:yes
- Age at study initiation: 42 -55 days
- Weight at study initiation: Females: 150.45-179.08 g, Males: 15392-17586 g
- Housing: Room D125 in the facility’s barrier system. Animals were housed in PC plastic cages (L46.0>- Diet: Animals were provided with sterilized diet with complete nutrition supplied by Beijing keaoxieli Feed Co., LTD. (Product License No: SCXK (jing) 2012-0004, Batch No. 16053123 and 16013213).
- Water: Drinking water is purified with HT—ROIOOO purity system
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.2—24.3 °C
- Humidity (%): 41 % - 73 %
- Photoperiod (hrs dark / hrs light): Controlled light cycle was 12 hour light/ 12 hour dark.

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
March 28, 2016 to June 28, 2016
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/L drinking water
Dose / conc.:
100 mg/L drinking water
Remarks:
Nominal
Dose / conc.:
250 mg/L drinking water
Remarks:
Nominal
Dose / conc.:
750 mg/L drinking water
Remarks:
Nominal
No. of animals per sex per dose:
Control (0 mg/L) 16 male, 16 female
Test item 100 mg/L 10 male, 10 female
Test item 250 mg/L 10 male, 10 female
Test item 750 mg/L 10 male, 10 female
Control animals:
yes
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a week at same time

BODY WEIGHT: Yes / No / Not specified
- Time schedule for examinations:

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The ration water consumption was determined once weekly at the same time as the water consumption determined, and added water volume was about 5OOmL and the bottles filled with water were weighed. The water was weighed again three days(72hd +/-1.5h) later as surplus water weight. Mean water consumption for one animal per day is calculated based on the above data. Calculation formulation: Mean daily water consumption (g) = [added water weight (g, including weight of water bottle)-surplus water weight (g, including weight of water bottle)]/6.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmic examinations were conducted on all animals prior to the dosing and at the end of treatment.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment period: No abnormal findings were observed in the hematological parameters in all treated animals. At the end of the recovery period: No abnormal findings were observed in the hematological parameters in all treated animals.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: not specified
- Animals fasted: Yes overnight before blood collection
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: Yes / No / Not specified
- Animals fasted: Yes / No / Not specified
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes / No / Not specified
- Time schedule for examinations:
- Dose groups that were examined:
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Yes
- Time schedule for examinations:
- How many animals:
- Dose groups that were examined:
- Parameters checked in table [No.?] were examined.

OTHER:
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see appendix 17.1— table 14, and individual values see appendix 17.2-table 12.)

HISTOPATHOLOGY: Yes (appendix 17.1-table 15, and individual values see appendix 17.2-table 13.)
Other examinations:
See results

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
During the whole test, one male animal was dead on Week 10th at the low dose (Animal ID1103) and showed ematiation and white opacity of eyeball. During the whole test, the symptoms noted were considered unrelated to treatment because they occurred in both the treatment and control groups or only appeared sporadically in low incidence throughout the study with no correlation to treatment.
For mortality results see appendix 17.1-table 1.
For clinical symptoms statistics results see appendix l7.l-table 2, and for individual symptoms results see appendix 17 .2—table 1.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
one male animal was dead on Week 10th at the low dose; For mortality results see appendix 17.1-table 1.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males: Animals at the dose of 750ppm showed a significant decrease in mean body weight from Week 1St to Week 2nd of the treatment period compared with the 0ppm group, and the body weight gains of the animals in the dose of 750ppm group recovered to normal from Week 3”. During the recovery period, there were no statistical significances compared the 750ppm dose group with the dose group of 0ppm.
Females: During the whole test, there were no statistical significances compared the treatment dose groups with the dose group of 0ppm. The results of the body weight measurement indicated that the body weight gains for males were inhibited by the test item at the dose of 750ppm at the beginning of the dosing via the drinking water for the decrease of the water intake.
For the mean body weight figure see appendix 17.1-Figures 1.1 and 1.2. Mean body weight variety statistic analysis results see appendix 17.1-table 4. For body weight individual values see appendix 17.2-table 2.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the treatment period, both male and female rats at 750ppm dose groups showed a significant decrease in mean food consumption on individual weeks compared with the control group. Both male and female rats at 250 or l00ppm dose groups showed a significant decrease in mean food consumption on individual weeks but there was no dose response relation with no toxicological meaning.
The results of the food consumption statistical analysis indicated that the amounts of food consumed of all animals at 750ppm dose groups were inhibited by the test item. For mean food consumption statistical analysis results see appendix l7.l-table 6. Food consumption individual values are shown in appendix 17.2-table 4.
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Males: On week 2“, water consumption was significantly decreased in all dose groups. Water consumption was significantly decreased at the dose of 250ppm and 750ppm fi'om week 2“. The total water consumption in 750ppm dose group for l to 13 week was significantly decreased compared with the control group.
Females: Water consumption was significantly decreased only on week 7th at the dose of 250ppm and 750ppm. But it happened accidently. The water consumption results indicated that male rats restricted their water intake in a dose- related fashion at the dose of 250ppm and 750ppm.
For mean water consumption statistical analysis results see appendix 17.1-table 5. Water consumption individual values are shown in appendix 17.2-table 3. For the
calculated amount(mg/kg per day) of the test item consumed on water consumption and body weight data statistical analysis results see appendix 17.1-table 5.
Ophthalmological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Ophthalmic examinations were conducted on all animals prior to the dosing and at the end of treatment. The results indicated that there were no treatment-related changes in the periocular area of eyes, conjunctiva, iris, cornea, optic disk and blood vessel of the eye
fundus. For the statistics results of ophthalmic examinations see appendix 17.1-tab1e 3.
Haematological findings:
no effects observed
Description (incidence and severity):
13.7.1 Hematological examination
At the end of the treatment period: No abnormal findings were observed in the hematological parameters in all treated animals.
At the end of the recovery period: No abnormal findings were observed in the hematological parameters in all treated animals.
The results of the hematological parameter’s statistical analysis indicated that there were no treatment—related findings during the study.
For hematological examination statistics results see appendix 17.1-table 8, and for individual values appendix 17.2—table 6.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The males at the dose of 750 ppm showed a significant increase in UREA and CREA compared with the Oppm dose group. Among males, GLU values were statistically significantly decreased at 75 Oppm dose, but with no toxicological meaning. Among females, no abnormal findings were observed in the biochemical parameters in all treated animals. At the end of the recovery period: No abnormal findings were observed in the biochemical parameters in all treated animals. The serum biochemical statistical analysis indicated that UREA and CREA values were increased by the test item at the dose of 750ppm for males. The above changes
were recovered to normal after stop ofdosing within the four weeks” recovery period. Serum biochemical examination statistics results were shown in appendix 17.1-table 9, and for individual values see appendix 17.2-table 7.
Urinalysis findings:
no effects observed
Description (incidence and severity):
The appearance of urine for all surviving animals were all normal. No toxicological meaning differences between control and treatment groups were found during the
whole test. For urinalysis statistics results see appendix 17.1-table 11, and for individual values see appendix 17.2-table 9.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: Low absolute values of epididymides weights were observed in male animals at all treatment groups compared with control group. Low relative values of epididymides weights were observed in male animals at 750 and l00ppm compared with control group. There was a statistically significant decrease in uterus absolute and relative weights at 250 and l00ppm dose for the females. statistically significant decrease in brain absolute weights at 750 ppm dose for the females. There was a statistically significant increase in heart relative weights at 750 ppm dose for the females. There was a statistically significant increase in thyroid relative weights at 750 ppm for the males. High absolute and relative values of thyroid weights were observed in female animals at 750 and 250ppm for the females. All organ weight changes noted above except thyroid were considered to be unrelated to treatment due to lack of dose responses. The results oforgan weight indicated that the absolute and relative values ofthyroid weights for the females were increased by the test item at the dose of 750 and 250ppm. For absolute organ weight and relative organ weight statistics results see appendix 17.1— -table 12, 13, and for individual absolute organ weights see appendix 17.2 —table 10, for individual relative organ weights see appendix 17.2 -table 11.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Gross necropsy and macroscopic observation At the end of the treatment period: One case of cerebrum malformation and small testicle was observed respectively in the 100ppm dose group. One case of lung nodule was observed in the 750ppm group. One case of kidney mass was observed in the l00ppm group. At the end of the recovery period: No abnormal findings were observed. Dead animals: No abnormal findings were in animal 1103.
The results indicated that there were no signs of toxicity related to the test item in gross necropsy and macroscopic observation during the whole test.
For statistics results of macroscopic observations see appendix 17.1— table 14, and individual values see appendix 17.2-table 12.
Neuropathological findings:
no effects observed
Description (incidence and severity):
The results of nerve fimction observation during the eleventh week of the treatment period indicated no neurotoxicity was found related to the test item. For neurotoxicity statistical analysis results see appendix 17.1-table 7. For individual values in nerve function observation see appendix 17.2-table 5.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: Low incidence of ventricle dilation in cerebrum, bone metaplasia in lung, focal necrosis in liver, edema in testis, atrophy in testis, mineralization of cecum, inflammatory cells infiltrate in heart, ectopic thymus in thyroid, nephroblastoma in kidney, dilatation of uterus were observed in the control group or treatment group. High incidence of inflammatory cells infiltrate in lung, vacuolar degeneration in liver were observed in both control and 75Oppm dose group.
There were no statistically significant differences and no dose-related responses by comparison of the ratios of the above abnormalities with the control in either group for both sexes. So no test item-related changes were observed in histopathology examinations. At the end of the recovery period: Low incidence of single cell necrosis in liver, vacuolar degeneration in liver, atrophy in testis, inflammatory cells infiltrate in pancreas were observed in the control and 750ppm dose group. High incidence of inflammatory cells infiltrate in lung and mineralization of colon were observed in both control and 750ppm dose group. There were no statistically significant differences by comparison of the ratios of the above abnormalities between the control and 750ppm dose group for both sexes. Dead animals: Focal necrosis in liver and murine progressive cardiomyopathy were observed in Animal 1103. Lung inflammatory cells infiltration was considered to be related to anesthesia and bloodletting. There were no statistically significant differences and no dose-related responses by comparison of the ratios of the above abnormalities between the control and 750ppm dose group for both sexes. So no test item—related changes were observed in histopathology examinations. Histopathology examination statistics results are shown in appendix 17.1-table 15, and individual values see appendix 17.2-table 13.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
13.7.3 Coagulation examination
At the end of the treatment period: The males at the 750 ppm dose level showed a significant increase in PT which was 5 % higher than control. The males at the 250 ppm dose level showed a significant increase in APTT which was 8 % higher than control. The above changes with no dose-relationship were considered to be marginal in nature. No abnormal differences were observed for the coagulation parameters in the female animals.
At the end of the recovery period: No abnormal differences were observed for the coagulation parameters in all treated animals.
The results of the coagulation parameter ’s statistical analysis indicated that there were no treatment—related findings during the study.
For coagulation examination statistics results see appendix 17.1-table 10, and individual for values appendix 17.2—table 8.
Details on results:
The results of the body weight measurement indicated that the body weight gains for males were inhibited by the test item at the dose of 750ppm at the beginning of the do sing via the drinking water. The water consumption results indicated that male rats restricted their water intake in a dose-related fashion at the dose of 250ppm and 750ppm, but it was not a adverse effect. Probably the drinking water was less tasty, especially at the highest dose level. The results of the food consumption statistical analysis indicated that the amounts of food consumed of all animals at 750ppm dose groups were inhibited by the test item. So the test item affected the food uptake, but there was no effect on body weight gain except in the male high dose group in only week 1 and 2 and there were no effects on histopathology. So the test item has an effect on the food consumption. However, this effect cannot be considered as an adverse efiect. The ophthalmic examinations results indicated that there were no treatment-related changes in the periocular area of eyes, conjunctiva, iris, cornea, opticdisk and blood vessel of the eye fundus. The results of nerve fimction observation during the eleventh week of the exposure indicated that no neurotoxicity was found related to the test item. The serum bio chemical statistical analysis indicated that UREA and CREA values were increased by the test item at the dose of 750ppm for males. The above changes were recovered to normal after stop of do sing within the four weeks’ recovery period. No toxicological meaning differences between control and treatment groups were found during the whole test in hematological parameter, coagulation parameter and urinalysis. The results oforgan weight indicated that the absolute and relative values of thyroid weights for the females were increased by the test item at the dose of 750 and 250ppm. But the microscopic slides of the thyroid did not show differences in histopathological observations between the dose groups. Finally, discontinuation of exposure to succinonitrile at the highest dose resulted in a decrease ofthe thyroid mass to control levels. Exposure to succinonitrile via drinking water resulted in an increase of the thyroid weight and this is an effect. This effect cannot be stated to be adverse in the absence of histopathological deviations in the thyroid compared to the control rats. No test item—related toxicity
changes were found in gross autopsy and macroscopic observation. Under the condition ofthis study, there were no toxicity pathology changes in SD rats with Succinonitrile. During the whole test, one male animal was dead on Week 10th at the low dose (Animal ID1103). To sum up the clinical observation, macroscopic
observation and the histopathology results, the cause of the death for Animal 1103 remained unknown. But there were no dose correlations and considered to be
unrelated to the test item.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
250 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios
Key result
Dose descriptor:
NOAEL
Effect level:
750 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
clinical biochemistry
organ weights and organ / body weight ratios

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
250 ppm
Organ:
thyroid gland

Applicant's summary and conclusion

Conclusions:
According to the above results, the no-observed-effect-level (NOEL) for Succinonitrile in repeated dose 90—day oral toxicity study in SD rats under the condition of the study was considered to be 250ppm for the males and females, and the calculated chemical intakes (mg/kg per day) of the test item were 24.8 +/- 3.5mg/kg/day for the females and 30.7 +/- 5.8mg/kg/day for the males..The no-observed—adverse-effect—level (NOAEL) for Succinonitrile was considered to be 750ppm for the males and females, and the calculated chemical intakes (mg/kg per day) of the test item were 70.0 +/- 8.8mg/kg/day for the males and 87.2 +/- 0.4mg/kg/day for the females. There was no significantly delayed occurrence of toxic effects during four weeks recovery period.
The results do not warrant a labelling for target organ toxicity by repeated exposure to succinonitrile.
Executive summary:

The study was performed to assess the toxicity of Succinonitrile in Sprague Dawley rats. The method was designed to meet the OECD Guideline for the Testing of Chemicals “Repeated Dose 90-Day Oral Toxicity in Rodents” (TG 408, adopted 1998).

Method: Oral (via the drinking water) was selected for administration for 3 months. There were four groups at different dose levels, including the control group (Oppm), the test item groups(100ppm, 250ppm and 750ppm). Animals in the control group received untreated drinking water. The drinking water at each dose group was analyzed for the first, middle and last preparation. For the first, middle, and last mixing periods, samples of drinking water at each dose level were taken from the

animal water bottles after the fourth day of preparation and analyzed for the test item. Clinical symptom observation, body weight measurement, the water consumption measurement, the food consumption measurement, ophthalmic examinations, nerve function observation and the clinical pathology including urinalysis, hematological, coagulation, blood biochemistry, necropsy and anatomic pathology and histopathology were carried out after the end of the dosing period and recovery period.

Results: The actual results for the analysis of the initial dosing formulation prepared on the first day were within i20% of the nominal concentration. The actual results

for the analysis of the dosing formulation from the water bottles were within +/-20% of the initial concentration. The results of the body weight measurement indicated that the body weight gains for males were inhibited by the test item at the dose of 750ppm at the beginning of the dosing via the drinking water. The water consumption results indicated that male rats restricted their water intake in a dose-related fashion at the dose of 250ppm and 750ppm, but it was not a adverse effect. Probably the drinking water was less tasty, especially at the highest dose level. The results of the food consumption statistical analysis indicated that the amounts of food consumed of all animals at 750ppm dose groups were inhibited by the test item. So the test item affected the food uptake, but there was no effect on body weight gain except in the male high dose group in only week 1 and 2 and there were no effects on histopathology. So the test item has an effect on the food consumption. However, this effect cannot be considered as an adverse effect. The ophthalmic examinations results indicated that there were no treatment—related changes in the periocular area of eyes, conjunctiva, iris, cornea, opticdisk and blood vessel of the eye fundus. The results of nerve function observation during the eleventh week of the exposure indicated that no neurotoxicity was found related to the test item. The serum biochemical statistical analysis indicated that UREA and CREA values were increased by the test item at the dose of 750ppm for males. The above changes were recovered to normal after stop of dosing within the four weeks” recovery period. No toxicological meaning differences between control and treatment groups were found during the whole test in hematological parameter, coagulation parameter and urinalysis. The results of organ weight indicated that the absolute and relative values of thyroid weights for the females were increased by the test item at the dose of 750 and 250ppm. But the microscopic slides of the thyroid did not show differences in histopathological observations between the dose groups. Finally, discontinuation of exposure to succinonitrile at the highest dose resulted in a decrease of the thyroid mass to control levels. Exposure to succinonitrile via drinking water resulted in an increase of the thyroid weight and this is an effect. This effect cannot be stated to be adverse in the absence of histopatho logical deviations in the thyroid compared to the control rats. No test item—related toxicity changes were found in gross autopsy and macroscopic observation. Under the condition of this study, there were no toxicity pathology changes in SD rats with Succinonitrile. During the whole test, one male animal was dead on Week 10th at the low dose (Animal ID1103). To sum up the clinical observation, macroscopic observation and the histopathology results, the cause of the death for Animal 1103 remained unknown. But there were no dose correlations and considered to be unrelated to the test item.

Conclusion: According to the above results, the no-observed-efi‘ect-level (NOEL) for Succinonitrile in repeated dose 90-day oral toxicity study in SD rats under the condition of the study was considered to be 250ppm for the males and females, and the calculated chemical intakes (mg/kg per day) of the test item were 24.8 +/-5mg/kg/day for the males and 30.7 +/-5.8mg/kg/day for the females. The no-observed—adverse—effect-1eve1 (NOAEL) for Succinonitrile was considered to be 750ppm for the males and females, and the calculated chemical intakes (mg/kg per day) of the test item were 70.0 +/-8.8mg/kg/day for the males and 87.2 +/-10.4mg/kg/day for the females. There was no significantly delayed occurrence of toxic efiects during four weeks recovery period.

The results do not warrant a labelling for target organ toxicity by repeated exposure to succinonitrile.