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Description of key information

Acute oral toxicity: rat (wistar) m/f (OECD guideline 420, GLP) Discriminating dose 500mg/kg bw; 3 deaths (1/5m/2/5f) at 2000mg/kg bw.
Acute Dermal Toxicity: rat (wistar) m/f (OECD guideline 402, GLP) Limit test 2000 mg/kg bw- no deaths, limited dermal irritation

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:
31 August - 4 October, 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD and in accordance with GLP. The study material is well characterized
Qualifier:
according to
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Qualifier:
according to
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
GLP compliance:
yes
Remarks:
Organisation for Economic Co-operation and Development, Principles on Good Laboratory Practice (revised 1997, ENV/MCJCHEM (98) 17)
Test type:
fixed dose procedure
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (U K) Ltd, Margate, Kent, U K
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: 228g to 246 g- males; 176 to 206 g-females
- Fasting period before study: overnight
- Housing: 5 to 6 per gender in solid-floor polypropylene cages furnished with woodflakes
- Diet (ad libitum): Rat and Mouse SQC Expanded Diet No.1, Special Diets Services Limi ted, Witha m, Essex, U K
- Water (ad libitum): mains drinking water
- Acclimation period: 5 days before

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19° to 25° C
- Humidity (%): 30-70 %
- Air changes (per hr): 15 approx
- Photoperiod (hrs dark / hrs light): 12 hours l ight (light cycle 06.00 to 18.00) and 12 hours darkness.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Doses:
2,000 mg/kg and 500 mg/kg bodyweight
No. of animals per sex per dose:
2 (preliminary study)
5 (Limit study)
Control animals:
no
Details on study design:
For the purpose of the study the test material was freshly prepared, as required, as a suspension in arachis oil BP (Batch number T27, Analytical Supplies Ltd., Little Eaton, Derby, UK). The preparations were mixed thoroughly using a Silverson Homogeniser and vortex mixer.

The absorption of AH18579 was not determined.

All animals were dosed once only by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its fasted bodyweight at the time of dosing.

At the end of the study the surviving animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormal ities was recorded . No tissues were retained .

Data evaluations included the relationship, if any, between the animals exposure to the test material and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects.
Statistics:
Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test material was made.
Preliminary study:
There were no deaths at dose levels of 500 and 2000 mg/kg bodyweight. Clinical observations noted at a dose level of 2000 mg/kg were hunched posture and pilo-erection.
Based on this information, dose levels of 2000 and 500 mg/kg bodyweight
were selected for the main study.
Sex:
male
Dose descriptor:
discriminating dose
Effect level:
ca. 500 mg/kg bw
Based on:
test mat.
Sex:
female
Dose descriptor:
discriminating dose
Effect level:
ca. 500 mg/kg bw
Based on:
test mat.
Mortality:
2,000 mg/kg: Three animals (one male and two females) were found dead 1, 2 or 5 days after dosing.
500 mg/kg: No deaths
Clinical signs:
2,000 mg/kg:
No signs of toxicity were noted during the day of dosing. Hunched posture was noted in four animals one day after dosing with pilo­ erection noted in three animals. Hunched posture was noted in seven animals on Day 2 and persisted in three animals on Days 3 and 4 and in one animal up to Day 7. Lethargy was noted in two animals on Day 3 and pilo-erection in one animal on Days 3 and 4. One male animal appeared normal throughout the study.


500 mg/kg:
There were no signs of toxicity noted during the study
Body weight:
2,000 mg/kg:
Surviving animals showed expected gains in bodyweight over the 14-day study per iod.

500 mg/kg:
All animals showed expected gains in bodyweight during the study.
Gross pathology:
2,000 mg/kg:
Abnormalities noted at necropsy of animals that died du ring the study were haemorrhagic or abnormal ly red lungs, dark liver or patchy pallor of the liver, dark kidneys, haemorrhagic gastric mucosa, haemorrhage or sloughing of the non-glandular epithelium of the stomach and haemorrhagic small and large intestines. Abnormalities noted at necropsy of two animals that were killed at the end of the study were thickened non-glandula r epithelium of the stomach and/or stomach adhered to the liver. No abnormalities were noted at necropsy of all other animals that were killed at the end of the study.


500 mg/kg:
No abnormalities were noted at necropsy
Interpretation of results:
Toxicity Category IV
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The acute oral median lethal dose (LD50) of the test material in the Wistar strain rat is considered to be between 500 and 2000 mg/kg.
Executive summary:

The discriminating dose in  the  main  study  was  established  at  500 mg/kg, with three animals dosed with 2000 mg/kg (1 male, 2 female) found dead 1,2 and 5 days after dosing. Application of the current classification critieria set out in OECD 420 (Fixed dose procedure) results in a CLP classification for Acute Oral toxicity of Category 4, with an ATE of 500 mg/kg.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
discriminating dose
Value:
500 mg/kg bw
Quality of whole database:
The study was conducted in 1999, in accordance with the OECD test method in force at that time, the study has recently been re-evaluated in accordance with modern classification criteria. The study is deemed to be a Klimisch 1 study

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:
21 January 2015 to 12 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Based on OECD test guideline and GLP compliant
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:
female
Details on test animals and environmental conditions:
Five male and five female Wistar (RccHanTM:WIST) strain rats were supplied by Harlan Laboratories UK Ltd., Oxon, UK. On receipt the animals were randomly allocated to cages. The females were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink-marking on the tail and a number written on a cage card. At the start of the study the animals weighed at least 200 g, and were 8 to 12 weeks of age. The weight variation did not exceed ±20% of the mean weight for each sex.

The animals were housed in suspended solid floor polypropylene cages furnished with woodflakes. The initial two animals were housed individually throughout the study. The further group of eight animals (four male and four female) were housed individually during the 24-Hour exposure period and in groups of four, by sex, for the remainder of the study. Free access to mains drinking water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study. The diet, drinking water and bedding were routinely analyzed and were considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 °C and 30 to 70% respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light (06:00 to 18:00) and 12 hours darkness.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
Type of coverage:
semiocclusive
Vehicle:
arachis oil
Details on dermal exposure:
The appropriate amount of test item, moistened with arachis oil BP, was applied as evenly as possible to an area of shorn skin (approximately 10% of the total body surface area). A piece of surgical gauze 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 that 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.

As no mortalities were noted a further group of animals (four males and four females) was similarly treated with the test item at a dose level of 2000 mg/kg body weight to give a total of five males and five females. 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. These animals were returned to group housing for the remainder of the test period.
Duration of exposure:
24 hours
Doses:
2000 mg/kg body weight
No. of animals per sex per dose:
5/sex/group
Control animals:
not specified
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed for deaths or overt signs of toxicity 30 minutes, 1, 2 and 4 hours after dosing and subsequently once daily for 14 days. Individual body weights were recorded prior to application of the test item on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: At the end of the study the animals were killed by cervical dislocation. All animals were subjected to gross necropsy. This consisted of an external examination and opening of the abdominal and thoracic cavities. The appearance of any macroscopic abnormalities was recorded. No tissues were retained.
- Other examinations performed: Clinical signs and body weight development were monitored during the study.
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
There were no deaths.
Clinical signs:
There were no signs of systemic toxicity.
Body weight:
All animals showed expected gains in body weight.
Gross pathology:
No abnormalities were noted at necropsy.
Other findings:
Dermal Irritation. Signs of dermal irritation noted were very slight to well-defined erythema, very slight edema, crust formation, light brown discoloration of the epidermis, small superficial scattered scabs and scab lifting to reveal glossy skin.
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.

The test item does not meet the criteria for classification according to CLP (1272/2008/EC).
Executive summary:

Introduction

The study was performed to assess the acute dermal toxicity of the test item in the Wistar strain rat.

Methods

Initially, two animals (one male and one female) 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 body weight. Based on the results of the initial test, a further group of eight animals (four males and four females) was similarly treated. 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.

Dermal Irritation. Signs of dermal irritation noted were very slight to well-defined erythema, very slight edema, crust formation, light brown discoloration of the epidermis, small superficial scattered scabs and scab lifting to reveal glossy skin.

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

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.

The test item does not meet the criteria for classification according to CLP (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
discriminating dose
Value:
500 mg/kg bw
Quality of whole database:
The study is contemporary, conducted in accordance with OECD and GLP guidelines and is deemed to be a Klimisch 1 study

Additional information

The available acute oral toxicity study was performed in 1999 in accordance with OECD Guideline 420 and GLP. The discriminating dose in  the  main  study  was  established  at  500 mg/kg, with three animals dosed with 2000 mg/kg (1 male, 2 female) found dead 1,2 and 5 days after dosing. Based upon the classification criteria in force at that time (Directive 67/548/EEC as adapted by Directive 93/21/EEC), the existing conclusion of the study director is that the LD50, based upon the fixed dose procedure is >2000mg/kg with a discriminating dose of 500mg/kg. Application of the current classification criteria in accordance with 1272/2008/EC results in a classification of Acute Toxicity 4 based upon the deaths observed in the female animals at 2000 mg/kg.

Acute dermal toxicity was assessed using OECD guideline 402 in accordance with GLP. A limit test at 2000 mg/kg bw was conducted which did not result in mortality or signs of systemic toxicity in the treated animals. Signs of dermal irritation noted in several animals included very slight to well-defined erythema, very slight edema, crust formation, light brown discoloration of the epidermis, small superficial scattered scabs and scab lifting to reveal glossy skin. The dermal reactions informed the need to assess the dermal irritancy potential in the integrated testing strategy.


Justification for selection of acute toxicity – oral endpoint
A single acute oral toxicity study is available, conducted in accordance with GLP and the OECD guideline in force at the time of testing.

Justification for selection of acute toxicity – dermal endpoint
A single acute dermal toxicity study is available, conducted in accordance with GLP and the OECD guideline currently in force.

Justification for classification or non-classification

The discriminating dose in  the acute oral  toxicity study  was  established  at  500 mg/kg, with three animals dosed with 2000 mg/kg (1 male, 2 female) found dead 1,2 and 5 days after dosing. Application of the current classification criteria set out in OECD 420 (Fixed dose procedure) results in a CLP classification for Acute Oral toxicity of Category 4, with an ATE of 500 mg/kg.

Given the lack of systemic toxicity observed in the acute dermal toxicity study, no classification for that endpoint is justified.