Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Endpoint summary

Description of key information

The test substance was determined to have a NOAEL for local effect in the stomach of 60 mg/kg bw/day for males and 20 mg/kg bw/day for females. The NOAEL for systemic toxicity was considered to be 60 mg/kg bw/day for males and females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-05-03 to 2016-09-20

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

(Crl:CD(SD)), SPF

Details on species / strain selection:

Sprague-Dawley rats are commonly used in both the general systemic toxicity and reproductive and developmental toxicity studies with a large historical control data base. In addition, the rat is a required species in the regulatory guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes (not stated directly but females were determined to have normal estrous cycle)
- Age at study initiation: male: 11 weeks; female: 12 weeks
- Weight at study initiation: male: 368.9 – 479.3 g; female: 224.7 – 274.6 g
- Fasting period before study: no
- Housing: Animals per cage: 1 (during the quarantine-acclimation period), 1 (during the dosing period), 1 male and 1 female (during the mating period), 1 female and neonates (during the lactation period); Stainless wire mesh cages, 260W×350D×210H (mm) and Polycarbonate cage 260W×420D×180H (mm)
- Diet: ad libitum, Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C)
- Water: ad libitum, tap water
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY:
The certificate of feed analysis was provided by the supplier and the results of feed analysis met the allowable standard of the test facility.
Public tap water was filtered and irradiated by ultraviolet light. Samples of drinking water are analyzed for specified microorganisms once a month and all environmental contaminants once a year according to the Regulation of Quality Criteria for Potable Water and Test. The results of water analysis met the allowable standard of the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 – 24.3
- Humidity (%): 48.0 – 69.7
- Air changes (per hr): 10 – 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Details on route of administration:

The oral route was chosen to assess the toxicity by oral exposure of the test substance.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of the test substance was weighed using an electronic balance (BP3100S, CP423S, Sartorius, Germany) and placed in a container. The test substance was mixed with a small amount of vehicle to dissolve using a magnetic stirrer, and then the vehicle was gradually added to yield the desired concentrations. The dosing formulations were stored in a refrigerator (3.9 – 5.4 °C). These dosing formulations were used within 7 days.

VEHICLE
- Justification for use and choice of vehicle: Through the preliminary solubility test to determine the solubility and dispersion characteristics of the test substance, corn oil was selected as the vehicle because the test substance was well dissolved in it.
- Concentration in vehicle: 0.2 and 200 mg/mL
- Amount of vehicle: 5 mL/kg
- Lot/batch no.: MKBV2080V

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses of the dosing formulations were conducted using a gas chromatography (GC-2010 series, Shimadzu Corp., Japan). Samples were taken three times from the middle of each dosing formulation prior to dosing and analyzed for verification of dose level concentration. The results of dose concentration analyses were determined to be 96.47 – 109.60 %. These results were within the acceptable limits (±15% of nominal values).

Duration of treatment / exposure:

Males of the main group were dosed for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating). Males and females of the recovery groups were dosed for 50 days. Females of the main group were dosed for 2 weeks prior to mating until Postpartum Day 13.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Low dose

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Mid dose

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

High dose

No. of animals per sex per dose:

Main groups: 12
Recovery groups: 6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In previously conducted 2-week repeated oral dose range finding study all animals were dead or moribund at 1000 mg/kg bw/day. Furthermore salivation and an increase in the relative liver weight were noted in females at 300 mg/kg bw/day. Therefore, the high dose level was selected at 200 mg/kg bw/day. Then, the mid and low dose levels were selected at 60 and 20 mg/kg bw/day, respectively.
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

No positive control was conducted.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the test and once a week throughout the dosing and recovery periods

BODY WEIGHT: Yes
- Time schedule for examinations: males of main group and males/females of recovery group: just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period and recovery period, the day prior to necropsy and on the day of necropsy (fasted body weights); females of main group: Body weights of females of the main group were recorded just prior to dosing on Day 1 (the first day of dosing), once a week throughout the dosing period, on Gestation Days 0, 7, 14 and 20, on Postpartum Days 0, 4 and 13, the day prior to necropsy and on the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No
Individual food consumption was calculated by subtracting the amount of residual feed from the amount presented.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes, as part of the detailed neurological examination

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy blood samples were collected from the abdominal aorta.
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, approximately 18 hours
- How many animals: all
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy blood samples were collected from the abdominal aorta.
- Animals fasted: Yes, approximately 18 hours
- How many animals: all
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: two days before necropsy
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes, cursory
- Time schedule for examinations: once before the test and once a week throughout the dosing and recovery periods
- Dose groups that were examined: all animals
- Battery of functions tested: motor activity, central and autonomic nervous system effects and behavior

NEUROBEHAVIOURAL EXAMINATION: Yes, detailed
- Time schedule for examinations: few days before necropsy
- Dose groups that were examined: six males and six females were randomly selected from the main groups in addition to all recovery animals
- Battery of functions tested: Pinna reflex, auditory (sound) reflex, corneal reflex, pupillary reflex, grip strength test, Rotarod test, spontaneous motor activity test

IMMUNOLOGY: Yes
- Time schedule for examinations: pups: on PND 4 and 13; adults: at termination
- How many animals: at least two pups per litter, all adult males and dams of the main group
- Dose groups that were examined: all
- Parameters examined: Total Thyroxine (T4) [ug/dL] Method: Chemiluminescent competitive immunoassay

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All surviving animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anesthesia. Complete gross postmortem examinations were conducted on all animals including the external surfaces and internal organs.

Organ weights:
The testis and epididymis of all adult males were weighed.
Six males and six females were randomly selected from the main group animals in addition to all recovery animals for necropsy. The organ weights from organs listed in Table No. 4 were determined.

HISTOPATHOLOGY: Yes. The testis, epididymis and eyes with optic nerves were fixed in Davidson’s fixative. All other tissues were preserved in 10% neutral buffered formalin. The thyroid from one male and one female pups per litter were preserved in 10% neutral buffered formalin on PND 13. Organs listed in Table No. 5 were prepared for histopathology. Examinations were conducted in six males and six females from the control, low, mid and high groups, for all gross, macroscopic lesions in all animals and in addition target organs noted at the high dose were examined for the recovery groups.

Statistics:

The statistical analysis of this study was conducted using the SAS program (SAS® 9.3, SAS Institute Inc., U.S.A.).
For the data including body weights, food consumption, thyroid hormone value, urine volume, hematology and clinical chemistry parameters, organ weights, sensory reactivity and motor activity, the Bartlett test was conducted to test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) test was employed on homogeneity, if significant (significance level: 0.05), followed by Dunnett’s t-test for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). Kruskal-Wallis test was employed on heterogeneity, and if significant (significance level: 0.05), Steel’s test was performed for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
For the data of the recovery groups, Folded-F test was employed to test for homogeneity of variance (significance level: 0.05, two-tailed). Student t-test was employed for homogeneity, but if overruled, Aspin-Welch t-test was applied (significance levels: 0.05 and 0.01, two-tailed).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the main groups, salivation was sporadically observed in six males at 200 mg/kg bw/day from Day 22 to the end of dosing. Also, salivation was observed temporarily in one female at 20 mg/kg bw/day and 2 females at 200 mg/kg bw/day.
In the recovery groups, salivation was often observed in three males and three females at 200 mg/kg bw/day from Day 14 to the end of dosing.
Salivation observed in both sexes at 200 mg/kg bw/day was considered to be a test substance-related effect.
No clinical signs were observed in males and females of the main and recovery groups in the detailed examinations.

Mortality:

no mortality observed

Description (incidence):

In the main and recovery groups, all animals survived the duration of the study. There was no effect on the mortality.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No test substance-related effects on the body weight changes were noted in both sexes at 20, 60 and 200 mg/kg bw/day in the main and recovery groups.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease in food consumption was noted in females at 20 mg/kg bw/day in the main group on Gestation Day 7. A statistically significant increase in food consumption was noted in females at 200 mg/kg bw/day in the recovery group on Day 50. However, these statistical significances were not considered to be a test substance-related change since the difference was of small magnitude and it was not related to the body weight changes.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test substance-related effects on pupillary reflex or corneal reflex were observed in animals of both sexes in the main and recovery groups when compared to the control group.

Haematological findings:

no effects observed

Description (incidence and severity):

No adverse effects were observed in any animal in the main and recovery groups.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No adverse effects were observed in any animal in the main and recovery groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

In males of the main and recovery groups, no effects were noted in any dosing group.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No test substance-related effects on auditory reflex or pinna reflex were observed in animals of both sexes in the main and recovery groups when compared to the control group.
In the main and recovery groups, there were no test substance-related effects in the grip strength test and spontaneous motor activity of both sexes when compared to the control group. A statistically significant increase was noted in rota rod test in males at 60 mg/kg bw/day when compared to control group. However, it was considered not to have a meaning of toxicological significance since it was without dose-dependency.
In the recovery groups, there were no test substance-related effects in the grip strength test, rota rod test and spontaneous motor activity when compared to the control group.

Immunological findings:

no effects observed

Description (incidence and severity):

There were no test substance-related changes in thyroid hormone (T4) level of F0 males and F1 pups at 20, 60 and 200 mg/kg bw/day.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test substance-related effects were noted in organ weights of males or females of any dose group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination at necropsy revealed test substance-related findings including focus on mucosa of the glandular stomach in one male at 200 mg/kg bw/day, one control female and females at 60 and 200 mg/kg bw/day, and polyp/thickening of mucosa in the forestomach in one female at 200 mg/kg bw/day.
At the end of the recovery period, these findings were not noted in any animal.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic changes observed in the stomach of both sexes were most likely to be test substance-related.
Epithelial hyperplasia/hyperkeratosis with inflammatory cell infiltration in the forestomach submucosa was noted in one female at 200 mg/kg bw/day. This finding corresponded to macroscopically observed polyp/thickening of the forestomach. Erosion on mucosa in the glandular stomach was observed in one male at 200 mg/kg bw/day and females at 60 and 200 mg/kg bw/day. This finding was in concordance with macroscopically observed focus on mucosa of the glandular stomach.
At the end of the recovery period, these findings were not observed in any animal.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The NOAEL for the test substance was determined to be 60 mg/kg bw/d for systemic toxicity after repeated application.

Executive summary:

An oral repeated dose toxicity study was conducted according to OECD 422 in rats. The test substance was administered via gavage to groups of 12 male and 12 female rats at concentrations of 0, 20, 60 and 200 mg/kg bw/d. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for thirteen days after delivery. Also, males and females of the recovery groups were dosed for 50 days. All males of the main group and all animals of the recovery groups survived the duration of the study. Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups. No test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, thyroid hormone (T4) of adult males and organ weights of animals of both sexes in the test substance-dosed groups. Focus of the glandular stomach was observed in one male at 200mg/kg bw/day and females at 60 and 200 mg/kg bw/day. Polyp/thickening of the forestomach mucosa was observed in one female at 200 mg/kg bw/day. Erosion on mucosa in the glandular stomach was observed in one male at 200 mg/kg bw/day and females at 60 and 200 mg/kg bw/day. Epithelial hyperplasia/hyperkeratosis with inflammatory cell infiltration in the forestomach submucosa was noted in one female at 200 mg/kg bw/day. Based on the results of this study, the NOAEL of the test substance for local effect in the stomach was considered to be 60 mg/kg bw/day for males and 20 mg/kg bw/day for females. The NOAEL for systemic toxicity was considered to be 60 mg/kg bw/day for males and females.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Guideline study

System:

gastrointestinal tract

Organ:

stomach

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

An oral repeated dose toxicity study was conducted according to OECD 422 in rats. The test substance was administered via gavage to groups of 12 male and 12 female rats at concentrations of 0, 20, 60 and 200 mg/kg bw/d. Males of the main group were dosed once daily for a total of 50 days (prior to mating for 2 weeks, during 2 weeks of mating and 22 days of post-mating), and females of the main group were dosed once daily for two weeks prior to mating, throughout gestation and for thirteen days after delivery. Also, males and females of the recovery groups were dosed for 50 days. All males of the main group and all animals of the recovery groups survived the duration of the study. Salivation was observed in males and females at 200 mg/kg bw/day in the main and recovery groups. No test substance-related adverse effects were noted in the results of detailed clinical signs, body weights, food consumption, estrous cycle, sensory function, motor activity, urinalysis, hematology, clinical chemistry, thyroid hormone (T4) of adult males and organ weights of animals of both sexes in the test substance-dosed groups. Focus of the glandular stomach was observed in one male at 200 mg/kg bw/day and females at 60 and 200 mg/kg bw/day. Polyp/thickening of the forestomach mucosa was observed in one female at 200 mg/kg bw/day. Erosion on mucosa in the glandular stomach was observed in one male at 200 mg/kg bw/day and females at 60 and 200 mg/kg bw/day. Epithelial hyperplasia/hyperkeratosis with inflammatory cell infiltration in the forestomach submucosa was noted in one female at 200 mg/kg bw/day. Based on the results of this study, the NOAEL of the test substance for local effect in the stomach was considered to be 60 mg/kg bw/day for males and 20 mg/kg bw/day for females. The NOAEL for systemic toxicity was considered to be 60 mg/kg bw/day for males and females.

 

A supporting study is available which was conducted to evaluate the potential toxicity of the test substance when administered once daily to 11-week-old male and 12-week-old female Sprague-Dawley rats by oral gavage for two weeks and to determine the dose levels for combined repeated dose toxicity study with the reproduction/developmental toxicity screening test. A total of 4 groups were designated as follows: Three groups were designated as Groups 2, 3 and 4 (low, middle and high dose groups, respectively) at dose levels of 100, 300 and 1000 mg/kg bw/day in addition to a control group (Group 1 corn oil). Each group consisted of 5 males and 5 females. Parameters were evaluated including clinical signs, body weights, food consumption, hematology, clinical chemistry, gross postmortem examinations and organ weights of selected tissues. Five males and two females were found dead at 1000 mg/kg bw/day.Three females were found moribund at 1000 mg/kg bw/day. Moribund or dead animals at 1000 mg/kg bw/day: A significant body weight loss was noted in both sexes on Day 4. Low values in RBC, HGB, HCT and WBC were noted in two moribund females. High values in ALT, AST, BUN, Crea, T-Chol and TG were noted in one moribund female. Black spot, black focus and/or perforation were observed in moribund or dead animals. Surviving animals: Salivation was observed in both sexes at 300 mg/kg bw/day. An increase in the relative liver weight was noted in males at 300 mg/kg bw/day. There were no treatment-related differences in body weights, food consumption, hematology, clinical chemistry and necropsy findings.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

Based on available data on repeated dose toxicity, the substance is considered to be not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the ninth time in Regulation (EU) No 2016/1179.