Benzene-1,3-dicarbonitrile

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2019 - 31 October 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

GLP study to current guideline

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzene-1,3-dicarbonitrile

EC Number:

210-933-7

EC Name:

Benzene-1,3-dicarbonitrile

Cas Number:

626-17-5

Molecular formula:

C8H4N2

IUPAC Name:

benzene-1,3-dicarbonitrile

Test material form:

solid: flakes

Details on test material:

Test substance name: Isophthalonitrile
Chemical name: 1,3-dicyanobenzene
CAS number: 626-17-5
EC number: 210-933-7
LOT number: JB1906150101
Purity: 99.8 % (w/w %)
Appearance: Off white solid flakes
Manufacture date: 15 June 2019
Expiry date:15 June 2020
Storage condition: Room temperature (15-25oC. below 70 RH%), protected from humidity

Specific details on test material used for the study:

Name: Isophthalonitrile
Chemical name: 1,3-dicyanobenzene
CAS number: 626-17-5
Batch/lot number: JB1906150101
Description: Off white solid flakes
Expiry date: 15 June 2020
Purity*: 99.8 % w/w
Storage conditions: Room temperature (15-25 ºC, below 70% relative humidity),
protected from humidity (tight closed container).
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.

Test animals

Species:

rat

Strain:

other: Han:WIST

Sex:

female

Details on test animals or test system and environmental conditions:

Species and strain: Han:WIST rats
Source: Toxi-Coop Zrt., H-1103 Budapest, Cserkesz utca 90.
Hygienic level at supplier: SPF
Hygienic level during
the study: Standard housing conditions
Justification of strain: The Wistar rat as a rodent is one of the standard strains for acute toxicity studies
Number of animals: 9 animals, 3 animals/group
Sex: Female, nulliparous and non-pregnant animals
Age of animals at dosing: Young adult rats, approx. 9-10 weeks old
Body weight range at dosing: 177-197 g. The maximum difference of individual animal weights from the mean of the treatment group did not exceed 20%.
Acclimatisation period: At least 14 days (14 / 15 / 19 days)

3.2.1. Husbandry

Animal health: Only healthy animals were used for the test. The health status was certified by the Veterinarian.
Number of animal room: R2.15/03
Housing: Group caging (3 animals/cage)
Cage type: T3H polycarbonate
Bedding and nesting: Aspen Bedding and sizzle pet nest material for Laboratory Animals (produced by Abedd Baltic Ltd. Kalnciems, Jelgavas, Latvia and LBS (Serving Biotechnology Ltd., Surrey RH6 0AH) were available to animals during the study.
Copies of the Certificates of Analyses are retained in the archives at NEXTREAT Laboratories.
Lighting period: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19 – 24 °C (target: 22 ± 3 °C)
Relative humidity: 35 – 73 % (target: 30 – 70 %)
Ventilation: 15-20 air exchanges/hour
Enrichment: Animals were housed by group to allow social interaction and with deep wood sawdust bedding to allow digging and other normal rodent activities.

The temperature and relative humidity were recorded twice daily during the study.

3.2.2. Food and water Supply

Animals received standard laboratory rat diet, ad libitum, and tap water from the municipal supply, as for human consumption from a 400 mL bottle, ad libitum. The food is not considered to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Details of the diet used in the study were as follows:

Name: Altromin 1324 Maintenance Diet – Rats/Mice
Manufacturer: Altromin Spezialfutter GmbH & Co. KG
Batch number: 201906031042
Expiry date: 03 March 2020

Copy of the certificate is archived with the raw data.

Water quality control analysis and microbiological assessment are performed once per year by the laboratory of Veszprém County Government Office, Department of Public Health (Veszprém Megyei Kormányhivatal Népegészségügyi Főosztály, H-8200 Veszprém, József A. u. 36., Hungary). The quality control results are retained in the archives of NEXTREAT Laboratories

3.2.3. Animal identification

Animals were individually identified by unique numbers written on the tail with an indelible pen. The numbers were given on the basis of NEXTREAT Laboratories' master file, for each animal allocated to the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered in 1% methyl cellulose solution, at a concentration of 30 or 200 mg/mL, at a dose volume of 10 mL/kg bw.

Doses:

300 mg/L and 2000 mg/L

No. of animals per sex per dose:

Three groups of three female rats were treated with the test item at a dose level of 300 (Groups 1 and 2) or 2000 mg/kg body weight (bw) (Group 3).

Control animals:

no

Details on study design:

Initially three animals were treated at the starting dose of 300 mg/kg bw (Group 1). As no mortality was observed in this group, a confirmatory group (Group 2) was treated at the same dose level. As no mortality was observed in this confirmatory group, a third group (Group 3) was treated at a higher dose level of 2000 mg/kg bw. One animal was found dead one day after the treatment. The remaining two animals were euthanized preterminally on the same day (Day 1), due to animal welfare reasons, in agreement with the Veterinary. As per the guideline, this is considered in the interpretation of the test results in the same way as the animals that died during the study. No further testing was required.

Results and discussion

Effect levels

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 - < 2 000 mg/kg bw

Based on:

test mat.

Mortality:

At the dose level of 300 mg/kg bw, no mortality was observed.
At the dose level of 2000 mg/kg bw, 1/3 animals was found dead and 2/3 animals were preterminally euthanized due to animal welfare reasons on Day 1.

Clinical signs:

Hunched back and piloerection were transiently seen in the animals dosed at 300 mg/kg bw,
2-4 hours after treatment. All animals became symptom-free 6 hours after treatment, until the end of the observation period.

The following symptoms were recorded in the animals after dosing at 2000 mg/kg bw: Slight to marked decreased activity (3/3), hunched back (3/3), incoordination (3/3), prone position (2/3), partially closed eyes (3/3), slight to moderate increased respiratory rate (3/3), and piloerection (2/3). Before their death/preterminal euthanasia, increased salivation (2/3) and clonic convulsion (3/3) were seen.

Body weight:

The body weights of the animals dosed at 2000 mg/kg bw were not evaluated because of the short observation period.
At 300 mg/kg bw, no treatment related changes were seen in the body weights.

Gross pathology:

In the terminally euthanized animals of the 300 mg/kg bw dose groups, there were no macroscopic changes seen at necropsy.
In the found dead or preterminally euthanized animals, those were dosed at 2000 mg/kg bw, discharge was seen around the mouth (2/3) and white material, presumably the test item, was present in the stomach (3/3).

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test item was found to be between 300 and 2000 mg/kg bw in female Han:WIST rats.
According to the GHS criteria, the test item can be ranked as "Category 4" for acute oral exposure.
The LD50 cut-off value is 500 mg/kg bw.

Executive summary:

The single-dose oral toxicity of the test item was performed according to the acute toxic class method (OECD 423 and Commission Regulation (EC) No 440/2008 of 30 May 2008, B.1.tris) in Han:WIST rats.

Three groups of three female rats were treated with the test item at a dose level of 300 (Groups 1 and 2) or 2000 mg/kg body weight (bw) (Group 3).

A single oral treatment was carried out by gavage for each animal after an overnight food withdrawal. Food was made available again 3 hours after the treatment. The test item was administered in 1% methyl cellulose solution, at a concentration of 30 or 200 mg/mL, at a dose volume of 10 mL/kg bw.

Initially three animals were treated at the starting dose of 300 mg/kg bw (Group 1). As no mortality was observed in this group, a confirmatory group (Group 2) was treated at the same dose level. As no mortality was observed in this confirmatory group, a third group (Group 3) was treated at a higher dose level of 2000 mg/kg bw. One animal was found dead one day after the treatment. The remaining two animals were euthanized preterminally on the same day (Day 1), due to animal welfare reasons, in agreement with the Veterinary. As per the guideline, this is considered in the interpretation of the test results in the same way as the animals that died during the study. No further testing was required.

Clinical observations were performed at once during the first 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter, where possible. Body weight was measured on Days -1 (prior to removal of food), 0 (prior to dosing), 7 and 14 (prior to necropsy), where possible. Terminal body weight of animals found dead or sacrificedin extremiswere also recorded. All animals were subjected to a necropsy and a macroscopic examination.