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Description of key information

LD50 > 2000 mg/kg in acute toxicity studies involving administration of test material to the Wistar rat via the oral route (OECD 420 and EU Method B.1 bis) and the dermal route (OECD 402 and EU Method B.3).

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:
08 September 2015 to 01 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
ANIMALS and ANIMAL HUSBANDRY
- Female Wistar rats were supplied by Envigo RMS UK Ltd, Oxon, UK.
- The animals were randomly allocated to cages on receipt.
- Females were nulliparous and non-pregnant.
- After an acclimatisation period of at least 5 days the animals were selected at random and given a unique number within the study by indelible ink-marking on the tail and the number was written on a cage card.
- At the start of the study the animals were 8 to 12 weeks of age.

- Body weight did not exceed ± 20 % of the mean body weight at the start of treatment.
- Animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- With the exception of an overnight fast immediately before dosing, and for approximately 3 to 4 hours after dosing, free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Envigo Research UK Ltd, Oxon, UK was allowed throughout the study.
- Diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect purpose or integrity of the study.
- Temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70 % respectively.
- Rate of air exchange was at least 15 changes per hour.
- Lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness.
- Animals were provided with environmental enrichment items considered not to contain any contaminant at a level that might affect the purpose or integrity of the study.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
TEST ITEM FORMULATION and EXPERIMENTAL PREPARATION
- For the purpose of the study, the test item was freshly prepared, as required, as a solution in arachis oil BP. This vehicle was chosen because the test material did not dissolve or suspend in distilled water. To aid dissolution the formulation was warmed in a water bath set at 40°C for upto 15 minutes
- The test item was formulated within 2 hours of being applied to the test system and it was assumed that the formulation was stable for that duration.
- No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and was reflected in the GLP compliance statement.
Doses:
- 2000 mg/kg (concentration 20mg/mL; dose volume 10 mL/kg)
No. of animals per sex per dose:
- Single female animal dosed at 2000 mg/kg
- In the absence of toxicity, a single female animal dosed at 2000 mg/kg
- In the absence of toxicity, an additional group of four female animals dosed at 2000 mg/kg
Control animals:
no
Details on study design:
- All animals were dosed once by gavage using a metal cannula attached to a graduated syringe.
- The volume administered to each animal was calculated according to the fasted body weight at the time of dosing.
- Treatment of animals was sequential.
- Sufficient time was allowed between each dose level to confirm the survival of the previously dosed animals.
- Clinical observations were made 0.5, 1, 2 and 4 hours after dosing and then daily for 14 days.
- Morbidity and motality checks were made twice daily.
- Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Animals were killed by cervical dislocation at the end of the observation period.
- All animals were subjected to gross necropsy consisting of external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded but no tissues were retained.
Statistics:
EVALUATION OF DATA
- The test item was evaluated according to Annex 3 of the OECD Guidelines for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed Dose Method (adopted 17 December 2001) as shown in the flow chart in Appendix 2 (attached).
- Evaluation of data included identification of the number of animals that died during the study (or that were killed for humane reasons), and determination of the nature, severity, onset and duration of toxic effects. If possible, the signs of evident toxicity were described. Evident toxicity refers to the toxic effects of sufficient severity that administration of the next higher dose level could result in development of severe signs of toxicity and probable mortality.
- Effects on body weights and abnormalities noted at necropsy were also identified.
- Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.
Preliminary study:
DOSE LEVEL 2000 mg/kg
- There was no mortality.
- No signs of systemic toxicity were noted during the observation period.
- The animal showed expected gains in body weight over the observation period.
- No abnormalities were noted at necropsy.
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
- There were no deaths.
Clinical signs:
- No signs of systemic toxicity were noted during the observation period.
Body weight:
- All animals showed expected gains in body weight over the observation period.
Gross pathology:
- No abnormalities were noted at necropsy.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was considered to be greater than 2000 mg/kg body weight.
Executive summary:

GUIDELINE

The study was designed to be compatible with OECD Guideline for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed Dose Method" (2001) and Method B1 bis Acute Toxicity (Oral) of Commission Regulation (EC) No 440/2008.

METHOD

Following a sighting test at a dose level of 2000 mg/kg, a further group of four fasted female animals were given a single oral dose of the test item at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

RESULTS

Mortality: There were no deaths.

Clinical observations: There were no signs of systemic toxicity.

Body weight: All animals showed expected gains in body weight.

Necropsy: No abnormalities were noted at necropsy.

CONCLUSION

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was considered to be greater than 2000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records
Reference
Endpoint:
acute toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
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:
09 September 2015 to 23 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Type of coverage:
semiocclusive
Vehicle:
unchanged (no vehicle)
Details on dermal exposure:
PROCEDURE
- On the day before treatment, the back and flanks of each animal were clipped free of hair.
- Using available information on the toxicity of the test item, five male and five female rats were treated with the test item at a dose level of 2000 mg/kg
- The calculated volume of test item, as received, waas applied as evenly as possible to an area of shorn skin (approximately 10 % of the total body surface area) using a graduated syringe.
- A piece of surgical guaze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage.
- The animals were caged individually throughout the study.
- Shortly after dosing the dressings were examined to ensure they were securely in place.
- After the 24 hour contact period the bandage was carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item.
- The animals were caged individually for the 24 hour exposure period.
- After the 24 hour contact period, the bandages were carefully removed and the treated skin and surrounding hair wiped with cotton wool moistened with arachis oil BP to remove any residual test item.
- The animals were returned to group housing for the remainder of the test period.
Duration of exposure:
24 hours
Doses:
2000mg/kg
No. of animals per sex per dose:
5 males and 5 females
Control animals:
no
Details on study design:
TEST ITEM FORMULATION AND EXPERIMENTAL PREPARATION
- The test item was weiged out weighed out according to each animals's individual body weight prior to application
- Absorption of the test item was not determined.
Statistics:
- Data evaluations included the relationsip, if any, between exposure of the animal to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, body weight changes, mortality and other toxicological effects.
- Using the mortality data obtained, and estimate of the acture dermal median lethal dose (LD50) of the test item was made.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
- There were no deaths
Clinical signs:
- No signs of systemic toxicity were noted during the observation period (see Table 1, attached).
Body weight:
- Individual body weights and body weight changes are shown in Table 4 (attached).
- Animals showed expected gains in body weight with the exception of two females, one showed no gain in body weight during the first & second week. The second female showed no gain in body weight during the first week and e expected gain in body weight took place during the second week.
Gross pathology:
- Individual necropsy findings are given in Table 5 (attached).
- No abnormalities were noted at necropsy.
Other findings:
DERMAL REACTIONS
- Individual dermal reactions are shown in Tables 2 and 3 (attached).
- No signs of dermal irritation were observed.
Interpretation of results:
not classified
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.
Executive summary:

GUIDELINE

Acute dermal toxicity was investigated in the Wistar rat using a method designed to be compatible with OECD Guidelines for the Testing of Chemicals No 402 "Acute Dermal Toxicity" (adopted 24 February 1987) and Method B.3 Acute Toxicity (Dermal) of Commission Regulation (EC) No 440/2008.

METHOD

Ten animals (five males and five females) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg bw. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

RESULTS

Mortality: No animal deaths took place during the study.

Clinical observations: No signs of systemic toxicity were observed.

Dermal irritation: No signs of dermal irritation were observed.

Body weight: . Animals showed expected gains in body weight with the exception of two females, one showed no gain in body weight during the first & second week. The second female showed no gain in body weight during the first week and expected gain in body weight took place during the second week

Necropsy: No abnormalities were noted at necropsy.

CONCLUSION

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Additional information

ORAL

The study was designed to be compatible with OECD Guideline for Testing of Chemicals No 420 "Acute Oral Toxicity - Fixed Dose Method" (2001) and Method B1 bis Acute Toxicity (Oral) of Commission Regulation (EC) No 440/2008.

Following a sighting test at a dose level of 2000 mg/kg, a further group of four fasted female animals were given a single oral dose of the test item at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

There were no deaths, there were no signs of systemic toxicity, all animals showed expected gains in body weight. No abnormalities were noted at necropsy.

The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was considered to be greater than 2000 mg/kg body weight

INHALATION

 

REACH Annex XIII, Section 8.5, Column 2, states that information on acute toxicity will be provided for the oral route plus at least one other route. The choice for the second route will depend on the nature of the substance and the likely route of human exposure. The test material is a liquid with a vapour pressure of 0.555 Pa at 25 °C, which makes generation of inhalable forms of the substance unlikely. As a result, it is considered that inhalation exposure will be low and the most likely route of exposure for workers and consumers is the dermal route. Testing for acute toxicity via the inhalation route is consequently not applicable.

DERMAL

Acute dermal toxicity was investigated in the Wistar rat using a method designed to be compatible withOECD Guidelines for the Testing of Chemicals No 402 "Acute Dermal Toxicity" (adopted 24 February 1987) and Method B.3 Acute Toxicity (Dermal) of Commission Regulation (EC) No 440/2008.

Ten animals (five males and five females) were given a single, 24 hour, semi-occluded dermal application of the test item to intact skin at a dose level of 2000 mg/kg bw. Clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

No animal deaths took place during the study. No signs of systemic toxicity were observed. No signs of dermal irritation were observed. Animals showed expected gains in body weight with the exception of two females, one showed no gain in body weight during the first & second week. The second female showed no gain in body weight during the first week and expected gain in body weight took place during the second week. No abnormalities were noted at necropsy.

The acute dermal median lethal dose (LD50) of the test item in the Wistar strain rat was found to be greater than 2000 mg/kg body weight.

 


Justification for selection of acute toxicity – oral endpoint
GLP Guideline Study

Justification for selection of acute toxicity – dermal endpoint
GLP Guideline study

Justification for classification or non-classification

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