5,7-di-tert-butyl-3-[4-(2-hydroxyethoxy)phenyl]-1-heteropolycycl-2(3H)-one

Endpoint summary

Description of key information

Acute oral toxicity: LD50 >2000 mg/kg bw, OECD 420, EU Method B1 bis, Sanders 2012.

Acute dermal toxicity: LD50 >2000 mg/kg bw, OECD 402, EU Method B3, Sanders 2013.

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:

02 February 2012 - 8 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 bis (Acute Oral Toxicity - Fixed Dose Procedure)

Deviations:

no

Principles of method if other than guideline:

Exception to GLP: No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of being applied to the test system; it is assumed that the formulation was stable for this duration. This exception is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Species:

rat

Strain:

other: Wistar (RccHanTM:WIST)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: 8 - 12 weeks.
- Weight at study initiation: The bodyweight variation did not exceed ± 20 % of the bodyweight of the initially dosed animal.
- Fasting period before study: Overnight fast immediately before dosing and for approximately three to four hours after dosing.
- Housing: The animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet (e.g. ad libitum): ad libitum 2014C Teklad Global Rodent diet (supplied by Harlan Laboratories UK Ltd., Oxon, UK)
- Water (e.g. ad libitum): free access to mains drinking water.
- Acclimation period: At least 5 days.

The diet, drinking water and bedding were routinely analysed and were considered not to contain any contaminants that would reasonably be expected to affect the purpose or integrity of the study.

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.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): At least 15 per hour.
- Photoperiod (hrs dark / hrs light): The lighting was controlled by a time switch to give twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.

IN-LIFE DATES: From: To: 2 February 2012 - 8 March 2012

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Experimental Preparation
For the purpose of the study the test item was freshly prepared, as required, as a suspension in arachis oil BP. Arachis oil BP was used because the test item did not dissolve/suspend in distilled water.

Procedure
In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.

A single animal was treated at a dose level of 300 mg/kg (concentration of 30 mg/mL and dose volume of 10 mL/kg).
In the absence of mortality or evident toxicity at a dose level of 300 mg/kg, an additional animal was treated at a dose level of 2000 mg/kg (concentration of 200 mg/mL and dose volume of 10 mL/kg).

In the absence of mortality or evident toxicity at a dose level of 2000 mg/kg, an additional group of 4 animals was treated at a dose level of 2000 mg/kg (concentration of 200 mg/mL and dose volume of 10 mL/kg).

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 the fasted bodyweight 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.

Doses:

Sighting test of 300 mg/kg bw
Limit test of 2000 mg/kg bw

No. of animals per sex per dose:

1 female in the sighting test
5 females in the limit test

Control animals:

no

Details on study design:

Clinical observations were made 0.5, 1, 2, and 4 hours after dosing and then daily for fourteen days.
Morbidity and mortality checks were made twice daily.

Individual bodyweights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.

At the end of the observation period 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.

Evaluation of Data:
Data evaluations included the relationship, if any, between the animals' exposure to the test item and the incidence and severity of all abnormalities including behavioural and clinical observations, gross lesions, bodyweight changes, mortality and any other toxicological effects. If possible the signs of evident toxicity were also identified. Evident toxicity is defined as the toxic effects which are of a severity such that administration at the next highest level could result in mortality.

Using the mortality data obtained, an estimate of the acute oral median lethal dose (LD50) of the test item was made.

Preliminary study:

Mortality
There was no mortality.

Clinical Observations
No signs of systemic toxicity were noted during the observation period.

Bodyweight
The animal showed expected gains in bodyweight over the observation period.

Necropsy
No abnormalities were noted at necropsy.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.

Clinical signs:

other: No signs of systemic toxicity were noted during 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 LD50 of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight.

Executive summary:

The LD50 of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight. The test was conducted in accordance with OECD Guidelines 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The key study was performed in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

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:

31 July 2013 - 14 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Principles of method if other than guideline:

On occasions, the humidity was outside the target range of 30 - 70 %. This was not felt to have affected the validity of the study.

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: RccHanTM:WIST
- Age at study initiation: 8 to 12 weeks old.
- Weight at study initiation: Animals weighed at least 200 g. The weight variation did not exceed ± 20 % of the mean weight for each sex. Males: 272 - 313 g, Females: 221 - 245 g
- Housing: Individually in suspended solid floor polypropylene cages during the exposure period, and then in groups of 5 per sex for the reminder of the study.
- Diet: Rodent diet available ad libitum.
- Water: drinking water available ad libitum.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 30 to 70 %
- Air changes (per hr): At least fifteen changes per hour.
- Photoperiod (hrs dark / hrs light): Twelve hours continuous light (06:00 to 18:00) and twelve hours darkness.
- Other: Animals provided with environmental enrichment items which did not contain any contaminant at a level that might affect the purpose or integrity of the study.

Type of coverage:

semiocclusive

Vehicle:

arachis oil

Details on dermal exposure:

TEST SITE
- Area of exposure: The backs and flanks of each animal (clipped).
- % coverage: Approximately 10 % of the total body surface area.
- Type of wrap if used: A piece of surgical gauze was placed over the treatment area and semi-occluded with a piece of self-adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: After removal of the bandage, the test site was wiped free of any excess test material using cotton wool moistened with arachis oil BP.
- Time after start of exposure: 24 hours.

TEST MATERIAL
- Amount applied: The test material was weighed out according to each animal's individual body weight.
- Application: An appropriate amount of the test material was moistened with arachis oil BP prior to application.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 male and 5 female per dose

Control animals:

no

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 ½, 1, 2 and 4 hours after dosing and subsequently once daily for fourteen days.
After removal of the dressings and subsequently once daily for fourteen days, the test sites were examined for evidence of primary irritation and scored according to the following the Draize (1977) scale, see Table 1. Any other skin reactions, if present were also recorded.
Individual body weights were recorded prior to application of the test material on Day 0 and on Days 7 and 14.
- Necropsy of survivors performed: Yes, 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.

Statistics:

Data evaluations included the relationship, if any, between the exposure of the animal to the test material and the incidence and severity of all abnormalities including behavioral and clinical observations, gross lesions, body weight changes, mortality and any other toxicological effects.
Using the mortality data obtained, an estimate of the acute dermal median lethal dose (LD50) of the test item was made.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths.

Clinical signs:

other: Clinical Signs: There were no signs of systemic toxicity.

Gross pathology:

No abnormalities were noted at necropsy.

Other findings:

Dermal Irritation: See tables 2 and 3.
Males: There were no signs of dermal irritation noted at the test site of one male. Very slight erythema (grade 1) was noted at the test sites of four males which persisted from 1 to 7 days post exposure.
Females: Well-defined erythema (grade 1 or 2) and very slight edema (grade 1) were noted at the test sites of all females. Erythema was observed from 1 day post exposure and persisted in 2 animals up to 13 days. Edemas were observed for the first 2 days only.
Other signs of dermal irritation noted at the test sites of females were crust formation, small superficial scattered scabs and scab lifting to reveal glossy skin. These observations were first noted on day 5 and persisted until termination on day 14.

Table 2: Male Local Reactions

Dose Level (mg/kg)	Animal No.	Observation	Days After Exposure
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	1-0	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-1	Erythema	1	1	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-2	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-3	Erythema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	1-4	Erythema	1	1	1	1	1	0	0	0	0	0	0	0	0	0
		Edema	0	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No reaction

Table 3: Female Local Reactions

Dose Level (mg/kg)	Animal No.	Observation	Days After Exposure
			1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	2-0	Erythema	2	2	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf	Cf	Cf	0	0	0	0	0	0	0
	2-1	Erythema	2	2	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	1	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf, Sg	Cf, Ss	Cf, Ss	Ss	Ss	Ss	Ss	Ss	Ss	Ss
	2-2	Erythema	2	2	2	2	2	2	2	1	1	1	1	1	1	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	Cf, Sg	Cf, Sg	Cf, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss, Sg	Ss
	2-3	Erythema	2	2	2	2	1	1	1	1	1	1	1	1	1	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	0-4	Erythema	2	1	1	1	1	1	1	0	0	0	0	0	0	0
		Edema	1	0	0	0	0	0	0	0	0	0	0	0	0	0
		Other	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0 = No reaction

Cf = Crust Formation

Ss = Small superficial scabs

Sg = Cabs lifting to reveal glossy skin

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the test, no mortality or systemic signs of toxicity were observed. Therefore the LD50 was determined to be > 2000 mg/kg bw. Furthermore only mild local irritation was observed at the test site.

Executive summary:

The acute toxicity of the test material was determined via the dermal route using male and female Wistar rats in a study performed under GLP conditions and in accordance with the standardised guidelines OECD 402 and EU Method B3.

A group of ten animals (five males and five females) was 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. Mortality, clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Under the conditions of the test no mortality or systemic signs of toxicity were observed. Animals displayed overall expected weight gain and no abnormalities were observed at necropsy. There were no signs of dermal irritation noted at the test site of one male. Very slight erythema was noted at the test sites of four males. Well-defined erythema and very slight edema were noted at the test sites of all females. Other signs of dermal irritation noted at the test sites of females were crust formation, small superficial scattered scabs and scab lifting to reveal glossy skin. Based on these observations the LD50 was determined to be > 2000 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Quality of whole database:

The key study was performed in line with GLP and standardised guidelines. It was assigned a reliability score of 1 in accordance with the criteria outlined in Klimisch (1997). It was therefore considered suitable to be the key study for this endpoint.

Additional information

Acute Oral Toxicity:

The acute oral toxicity of the test material was determined using female Wistar rats via the fixed dose method under GLP conditions and in accordance with the standardised guidelines OECD 420 and EU Method B.1 bis.

A single female was given a 300 mg/kg bw dose of the test material in arachis oil via oral gavage in a sighting test. Subsequently a group of five females were given dosed with the test material at 2000 mg/kg bw following the same procedure. Mortality, clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Under the conditions of the test no mortality or systemic signs of toxicity were observed. Animals displayed overall expected weight gain and no abnormalities were observed at necropsy. Based on these observations the LD50 was determined to be > 2000 mg/kg bw.

Acute Inhalation Toxicity:

In accordance with point 8.5.2, Column 2 (Specific rules for adaptation from Column 1), Annex VIII of Regulation (EC) No. 1907/2006, an acute inhalation study does not need to be performed as the substance has a low vapour pressure (<0.00043 Pa at 25 °C) and the use of this substance will not result in aerosols, particles or droplets of an inhalable size, so exposure to humans via the inhalatory route will be unlikely to occur. The acute toxicity endpoint has been addressed by assessing toxicity via the oral and dermal routes, which are more appropriate when considering the properties of this substance.

Acute Dermal Toxicity:

The acute toxicity of the test material was determined via the dermal route using male and female Wistar rats in a study performed under GLP conditions and in accordance with the standardised guidelines OECD 402 and EU Method B3.

A group of ten animals (five males and five females) was 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. Mortality, clinical signs and body weight development were monitored during the study. All animals were subjected to gross necropsy.

Under the conditions of the test no mortality or systemic signs of toxicity were observed. Animals displayed overall expected weight gain and no abnormalities were observed at necropsy. There were no signs of dermal irritation noted at the test site of one male. Very slight erythema was noted at the test sites of four males. Well-defined erythema and very slight edema were noted at the test sites of all females. Other signs of dermal irritation noted at the test sites of females were crust formation, small superficial scattered scabs and scab lifting to reveal glossy skin. Based on these observations the LD50 was determined to be > 2000 mg/kg bw.

Reliability

Both the key studies were performed in line with GLP and accepted standardised guidelines with a high standard of reporting. The studies were assigned a reliability score of 1 in accordance with the criteria outlined by Klimisch (1997) and considered suitable for assessment as an accurate reflection of the test material.

Justification for selection of acute toxicity – oral endpoint

Only one study available. Study was considered sufficiently reliable to address the acute oral toxicity endpoint on its own.

Justification for selection of acute toxicity – dermal endpoint

Only one study available. Study was considered sufficiently reliable to address the acute dermal toxicity endpoint on its own.

Justification for classification or non-classification

Acute Oral Toxicity:

In accordance with the criteria outline in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification for acute oral toxicity.

Acute Dermal Toxicity:

In accordance with the criteria outline in Regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification for acute dermal toxicity.