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Description of key information

Oral route: The acute oral median lethal dose (LD50) of the test item in female rats was demonstrated to be > 2000 mg/kg bw (OECD 420, EU Method B.1 bis, OPPTS 870.1100 and relevant Japanese guidelines)

 

Dermal route: The single dose acute dermal LD50 of test item was determined to be > 2000 mg/kg bw in male and female rats (OECD 402, EU Method B.3, OPPTS 870.1200 and relevant Japanese guidelines).

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:
15 August 2019 to 12 September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Version / remarks:
17 December 2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1100 (Acute Oral Toxicity)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau
Version / remarks:
24 November 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Remarks:
RccHan WIST albino
Sex:
female
Details on test animals or test system and environmental conditions:
ANIMAL INFORMATION
- Healthy nulliparous and non-pregnant female RccHan:WIST albino rats were obtained from Envigo RMS (UK) Ltd.
- The animals were allocated without conscious bias to cages within the treatment groups. They were housed in groups of one or four rats of the same sex.
- Each animal was identified uniquely within the study by tail marking. Each cage label was colour-coded and was identified uniquely with the study number, dose level and animal mark.
- The animals were allowed to acclimatise to the conditions described below for at least five days before treatment. For those animals selected for the study, body weights were in the range 161 to 170 g and they were approximately eight to twelve weeks of age prior to dosing (Day 1). The body weight variation did not exceed ± 20 % of the mean body weight of any previously treated animals.

ANIMAL CARE AND HUSBANDRY
- Animals were housed inside a limited access rodent facility (Building F21, Room 044/045). The facility was designed and operated to minimize the entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
- The animal room was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated. The temperature and relative humidity controls were set to maintain the range of 20 to 24 °C and 40 to 70 % respectively. Any minor deviations from these ranges would not have had an adverse effect on the animals and would not affect the integrity or validity of the study. Artificial lighting was controlled to give a cycle of 12 hours continuous light and 12 hours continuous dark per 24 hours. Environmental parameters are archived with the departmental raw data.
- Periodic checks were made on the number of air changes in the animal rooms. Temperature and humidity were monitored daily.
- Alarms were activated if there was any failure of the ventilation system, or temperature limits were exceeded. A stand-by electricity supply was available to be automatically brought into operation should the public supply fail.
- The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved softwood bark-free fiber bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
- The animals were allowed free access to a standard rodent diet (Teklad 2014C Diet), except for overnight prior to and approximately four hours after dosing. This diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Potable water taken from the public supply was freely available via polycarbonate bottles fitted with sipper tubes.
- Each cage of animals was provided with Aspen chew blocks or balls for environmental enrichment. Chew blocks or balls were provided throughout the study and were replaced when necessary. Each cage of animals was provided with a plastic shelter for environmental enrichment, which was replaced at the same time as the cages.
- Each batch of diet was analysed routinely by the supplier for various nutritional components and chemical and microbiological contaminants. Supplier’s analytical certificates were scrutinized and approved before any batch of diet was released for use. The quality of the water supply is governed by regulations published by the Department for Environment, Food and Rural Affairs. Certificates of analysis were received routinely from the water supplier. Certificates of analysis were received routinely from the supplier of the chew blocks or balls. Since the results of these various analyses did not provide evidence of contamination that might have prejudiced the study, they were not presented.
- No other specific contaminants that were likely to have been present in the diet or water were analysed, as none that may have interfered with or prejudiced the outcome of the study was known.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
FORMULATION
- The test item was formulated at concentrations of 30 and 200 mg/mL in the vehicle and administered at a volume of 10 mL/kg body weight.
- The absorption of the test item was not determined.
- Determination of the homogeneity, stability and purity of the test item or test item formulations were not undertaken as part of this study.
- Detailed records of test item usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion.
- The difference between these amounts was checked before the formulations were dispensed.

DOSE ADMINISTRATION
- The appropriate dose volume of the test item was administered to each rat by oral gavage using a plastic syringe and plastic catheter.
- A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly.
- Formulations were stirred before and throughout the dosing procedure.
Doses:
300mg/kg bw or 2000 mg/kg bw
No. of animals per sex per dose:
- One female dosed at 300 mg/kg bw
- Five females dosed at 2000 mg/kg bw
Control animals:
no
Details on study design:
- In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.
- A single female animal was treated at a dose level of 300 mg/kg (concentration 30 mg/mL; dose volume 10 mL/kg).
- A single female animal was treated at a dose level of 2000 mg/kg (concentration 200 mg/mL; dose volume 10 mL/kg).
- In the absence of mortality or toxicity at a dose level of 2000 mg/kg, an additional four animals were treated at a dose level of 2000 mg/kg (concentration 200 mg/mL; dose volume 10 mL/kg).
Statistics:
- Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
- There were no deaths during the study.
Clinical signs:
- Individual clinical observations are presented in Appendix 1 (attached).
- Clinical signs observed were piloerection, seen in four females dosed at 2000 mg/kg bw, first noted approximately three hours after dosing. Additionally, hunched posture was noted in two females dosed at 2000 mg/kg bw approximately six hours after dosing. Recovery of animals, as judged by external appearance and behaviour, was complete by Day 2. No clinical signs were seen in the animal dosed at 300 mg/kg bw or the remaining animal dosed at 2000 mg/kg bw.
Body weight:
- Individual body weight and body weight changes are given in Appendices 2 and 3 (attached).
- All animals were considered to have achieved satisfactory body weight gains throughout the study.
Gross pathology:
- Individual necropsy findings are given in Appendix 4 (attached).
- No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.
Interpretation of results:
GHS criteria not met
Conclusions:
The acute oral median lethal dose (LD50) of the test item in female rats was demonstrated to be > 2000 mg/kg bw.
Executive summary:

GUIDELINE

The investigation was conducted in compliance with the OECD Guideline for Testing of Chemicals No 420 “Acute Oral Toxicity – Fixed Dose Method” (17 December 2001), Method B.1 bis Acute Toxicity: fixed dose procedure (30 May 2008), EPA Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity EPA 712-C-02-190 (2002) and Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau (November 24, 2000).

 

METHODS

The purpose of the study was to assess the toxic potential of the test item following a single oral dose in the rat. Fasted female rats received a single oral gavage dose of the test item, formulated in corn oil,

at dose levels of 300 and 2000 mg/kg bw during sighting investigations). Based on the results of the sighting investigations a further four fasted females were similarly dosed at 2000 mg/kg bw. During the study, clinical condition, body weight and macropathology investigations were undertaken.

 

RESULTS

No unscheduled animal deaths took place during the study. Clinical signs observed were piloerection, seen in four females dosed at 2000 mg/kg bw, additionally hunched posture was noted in two females dosed at 2000 mg/kg bw. Recovery of animals, as judged by external appearance and behaviour, was complete by Day 2. No clinical signs were seen in any animal dosed at 300 mg/kg or the remaining animal dosed at 2000 mg/kg bw. All animals were considered to have achieved satisfactory body weight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

 

CONCLUSION

The acute oral median lethal dose (LD50) of the test item in female rats was demonstrated to be > 2000 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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:
05 December 2019 to 19 December 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity: Fixed Dose Procedure)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.1200 (Acute Dermal Toxicity)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF 12-Nousan-8147
Version / remarks:
2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
albino
Sex:
male/female
Details on test animals or test system and environmental conditions:
ANIMALS
- Source: Received from Charles River Laboratories on 21 November 2019.
- Number of animals: 10
- Sex: Five males and five females. Females assigned to the test were nulliparous and non-pregnant.
- Age: Young adult (10 weeks)
- Bodyweight: 274 to 327 g (males) and 221 to 246 g (females) at experimental start.

HUSBANDRY
- Housing: The animals were housed in caging which conforms to the size recommendations in the most recent Guide for the Care and Use of Laboratory Animals (Natl. Res. Council, 2011). Animals were group housed, except on the day of application, at which time they were singly housed until the animals were deemed acceptable, based on observations, to return to group housing. Enrichment (e.g. toy) was placed in each cage and litter was changed at least once per week.
- Animal Room Temperature and Relative Humidity Ranges: 21 to 24 °C and 41 to 56 % respectively.
- Animal Room Air Changes/Hour: 12. Airflow measurements are evaluated regularly and the records are kept on file at Product Safety Labs.
- Photoperiod: 12-hour light/dark cycle.
- Acclimation period: 14 days.
- Food: Food: Envigo Teklad Global 16 % Protein Rodent Diet #2016. The diet was available ad libitum.
- Water: Filtered tap water was supplied ad libitum.
- Contaminants: There were no known contaminants reasonably expected to be found in the food or water at levels which would have interfered with the results of this study. Analyses of the food and water are conducted regularly and the records are kept on file at Product Safety Labs.

IDENTIFICATION
- Cage: Each cage was identified with a cage card indicating at least the study number and identification and sex of the animal.
- Animal: A number was allocated to each rat on receipt and a stainless steel ear tag bearing this number was attached to the animal, This number, together with a sequential animal number assigned to study 51951, constituted unique identification. Only the sequential animal number was presented in the report.

PREPARATION AND SELECTION OF ANIMALS
- On the day prior to application, a group of animals was prepared by clipping the dorsal area and the trunk.
- After clipping and prior to application, the animals were examined for health, weighed (initial) and the skin checked for any abnormalities.
- Ten healthy, naïve rats (five males and five females; not previously tested) were selected for test.
Type of coverage:
occlusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
PREPARATION OF TEST SUBSTANCE
- The test substance was applied as received and mixed well prior to use.

DOSE CALCULATIONS
- Individual doses were calculated based on the initial body weights, taking into account the
density (determined by PSL) of the test substance.

APPLICATION OF THE TEST SUBSTANCE
- Test substance (2000 mg/kg bw) was applied evenly over a dose area of approximately 2
inches x 3 inches (approximately 10% of the body surface) and covered with a 2-inch x 3-inch, 4-ply gauze pad.
- The gauze pad and entire trunk of each animal were wrapped with 3-inch Durapore tape to avoid dislocation of the pad and to minimise loss of the test substance.
- The rats were then returned to their designated cages.
- The day of application was considered Day 0 of the study.
- After 24 hours of exposure to the test substance, the pads were removed and the test sites were gently cleansed with a 3 % soap solution followed by tap water and a clean paper towel to remove any residual test substance.

IN-LIFE OBSERVATIONS
- The animals were observed for mortality, signs of gross toxicity. and behavioral changes during
the first several hours after application, after patch removal, and then at least once daily thereafter
for 14 days.
- Observations included gross evaluation of skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous systems, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, and coma.

BODY WEIGHTS
- Individual body weights of the animals were recorded prior to test substance application (initial) and again on Days 7 and 14 (terminal).

NECROPSY
- All rats were euthanized via CO2 inhalation at the end of the 14-day observation period.
- Gross necropsies were performed on all animals.
- Tissues and organs of the thoracic and abdominal cavities were examined.
Duration of exposure:
24 hours
Doses:
Single dose of 2000 mg/kg bw
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Statistics:
STATISTICAL ANALYSIS
- Statistical analysis was limited to the calculation of the mean density value for dosing.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
- All animals survived test substance administration.
Clinical signs:
- Individual in-life observations are presented in Table 2 (attached).
- Dermal irritation was noted at the dose sites of six animals on Day 1. However, the animals recovered by Day 2, and along with the remaining animals, appeared active and healthy for the remainder of the 14-day observation period.
Body weight:
- Individual body weights and doses are presented in Table 1 (attached).
- All animals gained body weight during the study.
Gross pathology:
- Individual necropsy observations are presented in Table 3 (attached).
- No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the single dose acute dermal LD50 of test item is greater than 2000 mg/kg bw in male and female rats.
Executive summary:

GUIDELINE

The study was conducted in accordance with U.S. EPA Health Effects Test Guidelines, OPPTS 870.1200, OECD Guidelines for the Testing of Chemicals, Test No. 402, JMAFF 12-Nousan-8147 and the Official Journal of the European Union. Methods for the Determination of Toxicity and Other Health Effects, Part B.3 (Acute Toxicity Dermal), Council Regulation (EC) No. 440/2008.

 

METHODS

An acute dermal toxicity test was conducted with rats to determine the potential for test item to produce toxicity from a single topical application. Test substance (2000 mg/kg bw) was applied to the skin of ten healthy rats for 24 hours. The animals were observed for mortality, signs of gross toxicity, and behavioural changes at least once daily for 14 days. Body weights were recorded prior to application (initial) and again on Days 7 and 14 (terminal). Necropsies were performed on all animals at terminal sacrifice.

 

RESULTS

All animals survived test substance administration and gained body weight during the study. Dermal irritation was noted at the dose sites of six animals on Day 1. However, the animals recovered by Day 2, and along with the remaining animals, appeared active and healthy for the remainder of the 14-day observation period. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

 

CONCLUSION

Under the conditions of this study, the single dose acute dermal LD50 of test item is greater than 2000 mg/kg bw in male and female rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

Oral route

A key investigation was conducted in compliance with the OECD Guideline for Testing of Chemicals No 420 “Acute Oral Toxicity – Fixed Dose Method” (17 December 2001), Method B.1 bis Acute Toxicity: fixed dose procedure (30 May 2008), EPA Health Effects Test Guidelines OPPTS 870.1100 Acute Oral Toxicity EPA 712-C-02-190 (2002) and Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, Acute oral toxicity (2-1-1), 12 Nousan No 8147, Agricultural Production Bureau (November 24, 2000).

 

The purpose of the study was to assess the toxic potential of the test item following a single oral dose in the rat. Fasted female rats received a single oral gavage dose of the test item, formulated in corn oil,

at dose levels of 300 and 2000 mg/kg bw during sighting investigations). Based on the results of the sighting investigations a further four fasted females were similarly dosed at 2000 mg/kg bw. During the study, clinical condition, body weight and macropathology investigations were undertaken.

 

No unscheduled animal deaths took place during the study. Clinical signs observed were piloerection, seen in four females dosed at 2000 mg/kg bw, additionally hunched posture was noted in two females dosed at 2000 mg/kg bw. Recovery of animals, as judged by external appearance and behaviour, was complete by Day 2. No clinical signs were seen in any animal dosed at 300 mg/kg or the remaining animal dosed at 2000 mg/kg bw. All animals were considered to have achieved satisfactory body weight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

 

The acute oral median lethal dose (LD50) of the test item in female rats was demonstrated to be > 2000 mg/kg bw.

Inhalation route

The substance has been shown to have a high onset boiling point (178.7±0.5°C at 101.325 kPa) and the vapour pressure has been determined to be0.296Pa at 25 °C. It is therefore expected that inhalation exposure will be low under general use conditions at ambient temperature. In addition, lack of systemic toxicity when the substance is administered via the oral route suggests that determined vapour pressures of1.68 Pa at 65 °C and 2.35 Pa at 85 °Calso give no cause for concern. The inhalation route is therefore not the most applicable method of investigating acute toxicity.

Dermal route

The key study was conducted in accordance with U.S. EPA Health Effects Test Guidelines, OPPTS 870.1200, OECD Guidelines for the Testing of Chemicals, Test No. 402, JMAFF 12-Nousan-8147 and the Official Journal of the European Union. Methods for the Determination of Toxicity and Other Health Effects, Part B.3 (Acute Toxicity Dermal), Council Regulation (EC) No. 440/2008.

 

An acute dermal toxicity test was conducted with rats to determine the potential for test item to produce toxicity from a single topical application. Test substance (2000 mg/kg bw) was applied to the skin of ten healthy rats for 24 hours. The animals were observed for mortality, signs of gross toxicity, and behavioural changes at least once daily for 14 days. Body weights were recorded prior to application (initial) and again on Days 7 and 14 (terminal). Necropsies were performed on all animals at terminal sacrifice.

 

All animals survived test substance administration and gained body weight during the study. Dermal irritation was noted at the dose sites of six animals on Day 1. However, the animals recovered by Day 2, and along with the remaining animals, appeared active and healthy for the remainder of the 14-day observation period. No gross abnormalities were noted for any of the animals when necropsied at the conclusion of the 14-day observation period.

 

Under the conditions of this study, the single dose acute dermal LD50 of test item is greater than 2000 mg/kg bw in male and female rats.

Justification for classification or non-classification

No adverse effect was observed during investigation of acute toxicity via the oral or dermal routes in female rats and, based on determined vapour pressure of the substance, exposure via the inhalation route is not considered to be of significance to humans under general use conditions at ambient temperature. As such, classification in accordance with Regulation (EC) No 1272/2008 is not required.