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Description of key information

Acute oral toxicity

The acute oral toxicity was determined in a standard acute toxicity study according to OECD 401 and EC B.1 on male and female Wistar rats.

In conclusion, the combined LD50 on Wistar rats was found to be 1901 mg/kg; the LD50 for males 1736 mg/kg and for females 2026 mg/kg.

Acute inhalation toxicity:

Study was waived, because exposure of humans via inhalation is not likely taking into account vapor pressure and particle size of the substance. The test item is a solid at room temperature and usually handled as solid melt or in crystalline form. The content of inhalable particles of the crystalline material is very low. The particle size distribution of the crystalline material was found to be 125 - 500 μm. Less than 10% are <= 63 μm.

Acute dermal toxicity:

An acute dermal toxicity study was performed with the test item in Crl:(WI)BR rats, in accordance with testing methods EC B.3 and OECD 402. Under the experimental conditions the acute dermal LD50 value of the test item LZ 596 proved to be greater than 2000 mg/kg bw in male and female Crl:(WI)BR rats.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records
Reference
Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
May - June 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 401 (Acute Oral Toxicity)
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: P5256
- Aspect: off-white crystalline solid
- Batch no.: E 29.3.83

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Room temperature, protected from light
- Stability under test conditions: stable
- Expiry date: not given
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston Road, Margate, Kent, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6-10 weeks
- Weight at study initiation: males 143-193 g: females 127-177 g
- Fasting period before study: yes
- Housing: caged in groups of 4 by sex for the screening study and in groups of 5 by sex and dose group
- Diet (e.g. ad libitum): SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Special Diets Services Ltd., Stepfield, Witham, Essex, UK
- Water (e.g. ad libitum): tap water
- Acclimation period:5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21°C
- Humidity (%): 52-70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): Fluorescent lighting to give a cycle of 12 hours light and 12 hours darkness
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PRETEST:
Four groups, each of 2 fasted rats (1 male, 1 female), were dosed as follows:
- Dose level 50 mg/kg (Conc. 5 mg/ml) = dose volume 10.00 ml/kg
- Dose level 250 mg/kg (Conc. 25 mg/ml) = dose volume 10.00 ml/kg
- Dose level 1250 mg/kg (Conc. 125 mg/ml) = dose volume 10.00 ml/kg
- Dose level 5000 mg/kg (Conc. 500 mg/ml) = dose volume 10.00 ml/kg

MAIN STUDY:
Five groups, each of 10 fasted rats (5 males, 5 females), were dosed as follows:
- Dose level 1000 mg/kg (Conc. 200 mg/ml) = dose volume 5.00 ml/kg
- Dose level 1410 mg/kg (Conc. 200 mg/ml) = dose volume 7.05 ml/kg
- Dose level 1680 mg/kg (Conc. 200 mg/ml) = dose volume 8.40 ml/kg
- Dose level 2000 mg/kg (Conc. 200 mg/ml) = dose volume 10.00 ml/kg
- Dose level 2830 mg/kg (Conc. 200 mg/ml) = dose volume 14.15 ml/kg
Doses:
PRETEST:
- Dose level 50 mg/kg
- Dose level 250 mg/kg
- Dose level 1250 mg/kg
- Dose level 5000 mg/kg

MAIN STUDY:
- Dose level 1000 mg/kg
- Dose level 1410 mg/kg
- Dose level 1680 mg/kg
- Dose level 2000 mg/kg
- Dose level 2830 mg/kg
No. of animals per sex per dose:
PRETEST:
Four groups, each of 2 fasted rats (1 male, 1 female)

MAIN STUDY:
Five groups, each of 10 fasted rats (5 males, 5 females)
Control animals:
no
Details on study design:
APPEARANCE, BEHAVIOUR AND GENERAL OBSERVATIONS:
All animals were observed for overt signs of toxicity or behavioural change at 0.25, 1, 2 and 4 hours after treatment and subsequently once daily for 14 days. All gross or visible toxic or pharmacological effects were recorded.

BODY WEIGHTS:
Individual body weights were recorded on the day before treatment (day -1), on the day of treatment, 7 and 14 days after treatment and at death.

NECROPSY:
All animals were subjected to a gross necropsy examination. No tissues were retained. Animals surviving the 14 day observation period were killed by
exposure to high levels of carbon dioxide.
Statistics:
The acute oral median lethal dose (LDso) for combined male and female groups were calculated using a probit analysis (Finney, D.J. (1964), Statistical Method for Biological Assay, 2nd Edition, London, Charles Griffin). Separate LDso values were calculated for male and female animals. The mortalities did not allow the calculation of 95% fiducial limits or the production of a dose response curve.
Preliminary study:
Mortalities occured at dose levels of 1250 (50%) and 5000 mg/kg (100%). No mortlaity was noted at 50 and 250 mg/kg. Clinical signs were not determined.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
1 901 mg/kg bw
Based on:
test mat.
95% CL:
1 768 - 2 109
Key result
Sex:
male
Dose descriptor:
LD50
Effect level:
1 736 mg/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
2 026 mg/kg bw
Based on:
test mat.
Mortality:
A total of 18 (11 male, 7 female) of the 50 animals died during the study period. All deaths were noted within 5 days after treatment.
Clinical signs:
All animals treated with 1000 mg/kg appeared normal throughout the study period. All treated animals appeared normal during the day of dosing. Major signs of toxicity noted were lethargy and/or prostration, with occasional signs of piloerection, chromodacryorrhoea, hunched posture and staining around nose
and mouth.
Body weight:
With the exception of 1 animal treated with 1680 mg/kg all surviving animals showed body weight gains at the end of the study period. Animal 41 lost 36 g at termination.
Gross pathology:
Major pathological findings in animals dying during the study were associated with the stomach, which appeared distended. The livers were often pale with a speckled appearance and pale kidneys were also noted. With the exception of 1 animal treated with 1000 mg/kg which had dark lungs, and 2 animals treated with 1680 mg/kg which had pale livers, no other abnormalities were noted in animals necropsied at the end of the study period.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
The combined LD50 on Wistar rats was found to be 1901 mg/kg; the LD50 for males 1736 mg/kg and for females 2026 mg/kg.
Executive summary:

The acute oral toxicity was determined in a standard acute toxicity study according to OECD 401 and EC B.1 on male and female Wistar rats. Vehicle was corn oil. The appropriate dose levels were determined in a screening test on one male and female each, using doses of 50, 250, 1250 and 5000 mg/kg. Based on the pretest results, doses of 1000, 1410, 1680, 2000 and 2830 mg/kg were applied to 5 males and 5 females per group in the main study. A total of 18 (11 male, 7 female) of the 50 animals died during the study period. All deaths were noted within 5 days after treatment. All animals treated with 1000 mg/kg appeared normal throughout the study period. All treated animals appeared normal during the day of dosing. Major signs of toxicity noted were lethargy and/or prostration, with occasional signs of piloerection, chromodacryorrhoea, hunched posture and staining around nose and mouth. With the exception of 1 animal treated with 1680 mg/kg all surviving animals showed body weight gains at the end of the study period. Animal 41 lost 36 g at termination. Major pathological findings in animals dying during the study were associated with the stomach, which appeared distended. The livers were often pale with a speckled appearance and pale kidneys were also noted. With the exception of 1 animal treated with 1000 mg/kg which had dark lungs, and 2 animals treated with 1680 mg/kg which had pale livers, no other abnormalities were noted in animals necropsied at the end of the study period.

In conclusion, the combined LD50 on Wistar rats was found to be 1901 mg/kg; the LD50 for males 1736 mg/kg and for females 2026 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LD50
Value:
1 901 mg/kg bw
Quality of whole database:
Guideline study; Klimisch 1

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Link to relevant study records
Reference
Endpoint:
acute toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
- Species / Strain: Crl:(WI)BR rats
- Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
- Hygienic level: SPF at arrival and kept in a good conventional environment during the study
- Number of animals: preliminary study 2 female animals/dose / main study: 5 animals/dose
- Age of animals: Young adult rats, females were nulliparous and nonpregnant
- Housing: single housing (main study)
- Sex: female and male
- Body weight range: in preliminary study at starting: 252 - 285 g / in main study at starting: males 239 - 248 g / females 254 - 287 g
- Feeding: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany
- Water supply: tap water from municipal source
- Acclimatization time: in preliminary study: 47 days / in main study: 61 days in females and 5 days in malesTEST ANIMALS

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes: 10-15 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): Artificial light, from 6 a.m. to 6 p.m.
Type of coverage:
semiocclusive
Vehicle:
water
Remarks:
The test substance was moistened sufficiently with water to ensure good contact with the skin.
Details on dermal exposure:
The back of animals was shaven (approximately 10 % area of the total body surface) 24 hours prior to the treatment. The test item was applied in a single 2000 mg/kg bw dose uniformly over the shaved skin throughout a 24-hour exposure period. Sterile gauze pads were placed on the skin of rats. These gauzes were kept in contact with the skin by a patch with adhesive hypoallergenic plaster. The entire trunk of the animal was wrapped with semi
occlusive plastic wrap for 24 hours. At the end of the 24-hour exposure period any residual test item was removed using body temperature water
Duration of exposure:
24 hours
Doses:
- Preliminary test: 5, 50, 300, 2000 mg/kg
- Main test: 2000 mg/kg
No. of animals per sex per dose:
- Preliminary test: 2 females/dose
- Main test: 5 males and 5 females
Control animals:
no
Details on study design:
OBSERVATION:
A single administration was performed by dermal route and was followed by a 7-day observation period in preliminary study and 14-day observation period in main study.

MORTALITY:
Inspection for signs of morbidity and mortality were made twice daily at the beginning and end of the working day.

CLINICAL OBSERVATIONS:
Careful clinical observation was made at the following intervals: 1h, 5h after the treatment and once each day for 7 days thereafter in preliminary study and for 14 days thereafter in main study. Individual observations included the skin and fur, eyes and mucous membranes, and also respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern as well. Particular attention was directed to the observation of tremors,
convulsions, salivation, diarrhoea, lethargy, sleep and coma.

BODY WEIGHTS:
The body weight was recorded on day 0 (shortly before the treatment) in preliminary study and on day 0 (just before the treatment), on day 7 and on day 15 with a precision of 1 g in main study.

PATHOLOGY:
At the end of the observation period all rats of main study were sacrificed under isofluran anaesthesia. After examination of the external appearance, the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed, and any abnormality was recorded with details of its location, colour, shape and size. Animals of preliminary study were humanely sacrificed on Day 7.
Statistics:
no statistics perfomed
Preliminary study:
There were no deaths at 5, 50, 300 and 2000 mg/kg bw dose levels. Therefore, in the main test animals were administered the limit dose of 2000 mg/kg bw.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality were observed over a 14-day period following a 24-hour dermal exposure to the test item in male and female rats.
Clinical signs:
No behavioural changes, symptoms of dermal irritation and corrosion or signs of systemic toxity were noted during the study.
Body weight:
The mean body weight of the male animals corresponded to their species and age throughout the study. A body weight loss was observed in two females treated with 2000 mg/kg bw test item between Day 0 and Day 7. Both body weight losses were very slight (approx. 0.4 % in both cases) and the body weight of animals exceeded the original body weight by the end of study, thus it can be evaluated as an individual variation without toxicological meaning.
Gross pathology:
All animals survived until the scheduled necropsy on Day 15. Pale coloured kidneys as an internal necropsy finding were found in one male.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions, the acute dermal LD50 value of the test item proved to be greater than 2000 mg/kg bw in male and female WISTAR rats.
Executive summary:

An acute dermal toxicity study was performed with the test item in Crl:(WI)BR rats, in accordance with testing methods EC B.3 and OECD 402. A limit test was carried out. Single groups of male and female animals (n=5 animals/sex) were exposed to the test item at 2000 mg/kg bw by dermal route. The test item was applied in original form and left in contact with the skin for 24 hours, followed by a 14-day observation period. No mortality occurred during the study. Neither male nor female animals treated at 2000 mg/kg bw showed behavioural changes and no signs of systemic toxicity were observed during the study. No signs of irritation and corrosion were noted. The body weight development was undisturbed in male animals and slight body weight loss was observed in two females on first week, but it could not be evaluated as a toxic effect of test item. No macroscopic alterations of organs referred to the systemic toxic effect of the test item were seen during necropsy.

Under the experimental conditions the acute dermal LD50 value of the test item LZ 596 proved to be greater than 2000 mg/kg bw in male and female Crl:(WI)BR rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 mg/kg bw
Quality of whole database:
Guideline study; Klimisch 1

Additional information

Justification for classification or non-classification

According to the GHS criteria, the test item needs to be classified as "Acute tox. Cat. 4" for acute oral exposure.