Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile

Endpoint summary

Description of key information

OECD 423: Acute Oral LD₅₀(Rat) > 2000 mg/Kg bw

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:

04 June 2018 - 28 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

17 Dec 2001

Deviations:

yes

Remarks:

Deviations were considered not to adversely affect the results of or integrity of the study.

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

20 May 2008

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

712-C-98-190 (1998)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Test Item: Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene1-carbonitrile
Public name: Azuril
EC number: 915-371-2
CAS number: n/a
Batch/lot number: A170421E
Description: Clear, pale yellow liquid
Expiry date: 06 June 2019
Purity: 99.35%

Storage conditions: Room temperature (15-25 °C, ≤70 RH%), under inert gas, protected from humidity (tight closed container)

Identification of test item: The identification of the test item was made in the Pharmacy of Citoxlab Hungary Ltd. on the basis of the information provided by Sponsor.

Treatment of test material prior to testing: The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of Citoxlab Hungary Ltd. on the day of administration. The formulation container was magnetic stirred continuously up to the end of dose administration procedures.

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Crl:WI Wistar rats
Sex: female
Females nulliparous and non-pregnant: yes
Age at study initiation: 8 weeks
Weight at study initiation: 202-210 g
Fasting period before study: overnight, food returned 3 h after treatment
Diet: ad libitum - ssniff® SM R/M Autoclavable complete diet for rats and mice – breeding and maintenance
Water: ad libitum – tap water used for human consumption
Acclimation period: 7-8 days
Housing: group caging (3 rats/cage)
Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany

ENVIRONMENTAL CONDITIONS
Temperature (°C): 20.2 – 25.0
Humidity (%): 40 – 79
Air changes (per hr): 15-20
Photoperiod (hrs dark / hrs light): 12/12

Treatment: 05/06 June 2018
Observation: 05/06 June – 19 June 2018
Necropsy: 19/20 June 2018

Route of administration:

oral: gavage

Vehicle:

other: PEG 400 (polyethylene glycol 400)

Details on oral exposure:

VEHICLE: PEG 400
- Concentration in vehicle: 200 mg/mL of test material in the vehicle
- Justification for choice of vehicle: The selection of PEG 400 was made during trial formulations with the test item. Vehicles tested in trial formulations were distilled water, 1% methyl cellulose solution, corn oil, PEG 400 and propylene glycol. Corn oil and PEG 400 resulted in visibly clear solutions, other formulations were not visibly homogenous. PEG 400 (polyethylene glycol 400) was selected for this study and for the upcoming repeated dose rat studies, because it is more adapted to the chemical analytical methods to be used. In addition, PEG 400 is frequently used vehicle in rodent toxicological studies due to its well known characteristics and low toxicity (Gad et al. 2016. Int J Toxicol 35, 95-178).
- Lot/batch no: A0378559
- Purity: NA
- Expiry Date: 30 November 2021
- Dose volume: 10 mL/kg bw

CLASS METHOD
- Three female rats were treated with the limit dose 2000 mg/kg bw of the test item. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw. As no mortality was observed in this second dose group, further testing was not required according to the test guidelines. The dose of 2000 mg/kg bw was considered acceptable.

Doses:

Step 1: 2000 mg/kg bw
Step 2: 2000 mg/kg bw

No. of animals per sex per dose:

Step 1: 3
Step 2: 3

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily
- Necropsy of survivors performed: yes
- Other examinations performed: Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. Body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), weekly thereafter and at necropsy (Day 14).

Preliminary study:

Three female animals were treated with 2000 mg/kg bw of the test item (step 1). No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw (step 2).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

The test item did not cause mortality.

Clinical signs:

other: No evidence of adverse clinical signs observed.

Gross pathology:

No evidence of macroscopic changes observed during necropsy.

Interpretation of results:

Category 5 based on GHS criteria

Conclusions:

The LD50 for acute oral toxicity of the test material Azuril (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex3-ene-1-carbonitrile and 4-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile) was found to be above 2000 mg/kg bw in female Crl:WI Wistar rats. According to the GHS criteria, classification of the test item can be ranked as "Category 5 or Unclassified" for acute oral exposure.

Executive summary:

The aim of this study was to evaluate the acute oral (gavage) toxicity potential of the test material Azuril [Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex3-ene-1-carbonitrile and 4-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile].

The limit test using the dose of 2000 mg/kg bw was performed with totally six female Crl:WI Wistar rats. For the first step, three rats were treated without mortality. Therefore, a second group of three female rats was treated at the same dose level. No further testing was required, as no mortality was observed at 2000 mg/kg bw. PEG 400 (polyethylene glycol 400) was selected as a vehicle for this study.

Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weights were measured on days -1, 0, 7 and before necropsy (day 14). No mortality was observed, and body weight values were in the normal ranges. No evidence of the macroscopic changes was observed during the necropsy.

In conclusion, the acute oral LD50 value of the test item, Azuril (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) was found to be above 2000 mg/kg bw in female Crl:WI Wistar rats.

According to the GHS criteria, classification of the test item can be ranked as "Category 5 or Unclassified" for acute oral exposure.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Acute toxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The acute oral (gavage) toxicity potential of the test material Azuril (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex3-ene-1-carbonitrile and 4-(4-methyl-3pentenyl)cyclohex-3-ene-1-carbonitrile) was evaluated according to the OECD 423 test guideline and under GLP conditions.

 

Two groups of three female Crl:WI rats were treated with the test item at a dose level of 2000 mg/kg body weight. Clinical observations were performed at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. Body weights were measured on Days -1, 0, 7 and before necropsy (Day 14). The body weight values were in the normal ranges and there was no evidence of the macroscopic changes observed. No further testing was required,

as no mortality was observed at 2000 mg/kg bw. PEG 400 (polyethylene glycol 400) was used as a vehicle.

 

In conclusion, the acute oral LD50 value of the test item, Azuril (Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile) was found to be above 2000 mg/kg bw in female Crl:WI Wistar rats. According the GHS criteria, classification of the test item can be ranked as "Category 5 or Unclassified" for acute oral exposure.

Justification for classification or non-classification

Reaction mass of 3-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile and 4-(4-methyl-3-pentenyl)cyclohex-3-ene-1-carbonitrile (Azuril) does not meet the criteria to be classified for acute oral toxicity under EU Regulation (EC) No 1272/2008 (CLP). However, according to the GHS criteria, based on an oral LD₅₀of >2000 mg/Kg bw, the test material can be classified as "Category 5" or “Unclassified” for acute oral exposure.