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Description of key information

- Acute oral toxicity, rat (OECD 420): LD50 (m/f) > 2000 mg/kg bw

- Acute dermal toxicity, rat (OECD 402): LD50 (m/f) > 2000 mg/kg bw

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:
04 Oct - 07 Nov 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
EU Method B.1 (Acute Toxicity (Oral))
Version / remarks:
Annex to Directive 92/69/EEC (OJ No. L383A, 29,12,92), Part B, Method B.1)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Version / remarks:
Adopted 17 Dec 2001.
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Good Laboratory Practice, The United Kingdom Compliance Programme, Department of Health and Social Security 1986 and subsequent revision, Department of Health, UK
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Hsd/Ola: Sprague-Dawley (CD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Ltd., Bicester, Oxon, UK
- Females: yes
- Age at study initiation: 4 - 7 wks
- Weight at study initiation: 94 - 100 g (males range), 99 - 105 g (females range)
- Fasting period before study: animals were fasted overnight prior to and approximately 4 hours after dosing
- Housing: groups of up to 5 rats of the same sex per cage, in metal cages with mesh floors
- Diet: SDS LAD 1, ad libitum
- Water: drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 24 Oct 1995 To: 07 Nov 1995
Route of administration:
oral: gavage
Vehicle:
methylcellulose
Remarks:
1%, aqueous
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 20% w/v
- Amount of vehicle (if gavage): 10 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

CLASS METHOD
- Rationale for the selection of the starting dose: limit test in compliance with Test Guideline OECD420.
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were observed for mortality, morbidity and clinical signs soon after dosing and at frequent intervals on Day 1, then twice daily for 14 days. The bodyweight of each rat was recorded on Day 1 (prior to dosing), 8 and 15.
- Necropsy of survivors performed: yes
Statistics:
Individual weekly bodyweight changes and group mean bodyweights.
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Mortality:
No mortality occurred during the study period.
Clinical signs:
2000 mg/kg bw: piloerection was observed in all rats within 5 min of dosing, recovery was complete in all instances by three hours after dosing. There were no other clinical signs.
Body weight:
Slightly low bodyweight gains were recorded for two male rats on both Day 8 and 15, all other rats achieved satisfactory bodyweight gains throughout the study.

Main Study – Individual Bodyweights and % Weekly Bodyweight Increases, and Mortality

Dose Animal Bodyweight (g) % Increase Mortality

DAY1 DAY8 DAY15 DAY8 DAY15

2000 mg/kg 1 M 97 148 188 51 40 0/5
2 M 97 166 217 69 51
3 M 97 163 220 66 57
4 M 94 152 195 58 43
5 M 100 150 176 50 26
MEAN 97 156 199 -- --

2000 mg/kg 6 F 101 154 181 53 27 0/5
7 F 105 158 179 53 21
8 F 99 137 155 38 18
9 F 104 140 155 36 15
10 F 101 148 181 47 33
MEAN 102 147 170 -- --
Gross pathology:
No macroscopic abnormalities were observed for animals killed on Day 15.

 

Dose

[mg/kg bw]

Mortality

Clinical Signs

N

N

Males

2000

0/5

5/5

Females

2000

0/5

5/5

N = Number of animals dosed

Clinical Signs = Piloerection was observed in all rats, completely resolving in all instances by 3 hours after dosing

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
The acute lethal oral dose (LD50) to rats of AF-317 was found to be greater than 2000 mg/kg bw.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VII, 8.5, of Regulation (EC) No 1907/2006.

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:
15 Apr - 29 Apr 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no information on test substance formulation
Qualifier:
according to guideline
Guideline:
OECD Guideline 402 (Acute Dermal Toxicity)
Version / remarks:
adopted 1987
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.3 (Acute Toxicity (Dermal))
Version / remarks:
Annex V to Directive 92/69/EEC (OJ No. L383A, 29,12,92) , Part B , Methods for the determination
of Toxicity , B.3
Deviations:
no
GLP compliance:
yes
Test type:
standard acute method
Limit test:
yes
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Expiration date of the lot/batch: 03 July 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark


Species:
rat
Strain:
Wistar
Remarks:
Wistar strain Crl : (WI) BR (outbred, SPF-Quality)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Males: 328 +/- 22 g (mean +/- SD); Females: 222 +/- 12 g (mean +/- SD). Body weight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: No
- Housing: Individually housed in labelled poycarbonate cages containing purified sawdust as bedding material (Woody SPF, supplied by B.M.I., Helmond, The Netherlands)
- Diet: Standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium), ad libitum
- Water: Free access to tap water , ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): Approximately 15
- Photoperiod (hrs dark / hrs light): 12 / 12
Type of coverage:
occlusive
Vehicle:
propylene glycol
Details on dermal exposure:
TEST SITE
- Area of exposure: Back (one day before exposure an area of approx. 5 x 7 cm was clipped)
- % coverage: Approx. 10% of the total body surface, i. e. approx. 25 square cm for males and 18 square cm for females
- Type of wrap if used: Nonwoven swab ( Dispomed® L) covered with aluminium foil and Coban flexible bandage. A piece of Micropore tape was additionally used for fixation of the bandages in females only. Suppliers: Lohmann, Neuwied, Germany (Dispomed) and 3M, St. Paul, USA (Coban & Micropore).

REMOVAL OF TEST SUBSTANCE
- Washing: Yes, with a tissue moistened with tap water
- Time after start of exposure: 24 h

TEST MATERIAL
- Amount applied: 10 mL/kg bw

Duration of exposure:
24 h
Doses:
2000 mg/kg bw
No. of animals per sex per dose:
5
Control animals:
not required
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality / Viability: twice daily, body weight: Day 1 (pre-administration), 8 and 15, clinical signs: at periodic intervals on the day of dosing (Day 1) and once daily thereafter, until Day 15
- Necropsy of survivors performed: Yes
Statistics:
No
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
> 2 000 mg/kg bw
Based on:
test mat.
Remarks on result:
other: No mortality
Mortality:
No mortality occurred.
Clinical signs:
Red staining of the head, neck and / or periorbital region was noted in three animals on Day 2. Erythema (Grade 1), scales and scabs were seen in the treated skin-area among some animals between Days 3 and 6. Brown staining of the skin on the back by the test substance was noted in two females on Day 2.
Body weight:
The changes noted in mean body weight gain in males and females were within the range expected for rats used in this type of study and were therefore considered not indicative of toxicity.
Gross pathology:
Pelvic dilatation in the right kidney, found in one female at macroscopic post mortem examination, is commonly noted among rats of this age and strain and was therefore not considered to be toxicologically significant. No further abnormalities were noted among the animals.

Table 1. Mortality and clinical signs in the acute dermal toxicity study.

Dose

[mg/kg bw]

Mortality

Clinical signs

N*

N*

Males

2000

0/5

3/5

Females

2000

0/5

4/5

*N= Number of animals / number of animals used

Clinical signs: Red staining of the head, neck and / or periorbital region was noted in three animals (2 males and 1 female) on Day 2. Erythema (Grade 1), scales and scabs were seen in the treated skin-area among some animals (2 males and 3 females) between Days 3 and 6. Brown staining of the skin on the back by the test substance was noted in two females on Day 2.

Interpretation of results:
other: CLP/EU GHS criteria are not met, no classification required according to Regulation (EC) No 1272/2008
Conclusions:
The acute lethal dermal dose (LD50) of AF-317 to rats was found to be greater than 2000 mg/kg bw.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
The available information comprises an adequate and reliable study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.5, of Regulation (EC) No 1907/2006.

Additional information

Acute oral toxicity

A reliable acute toxicity study conducted under GLP with 2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone (CAS 903-19-5) in five male and female Sprague-Dawley rats according to OECD guideline 420 is available (Key, 1996). In this study the test substance diluted in methylcellulose was orally administered via gavage at a dose of 2000 mg/kg bw, and thereafter the rats were observed for 14 days. No mortality occurred during the study period. Piloerection was observed in all rats within 5 min of dosing, while the recovery was complete in all instances by 3 hours after dosing. Slightly low bodyweight gains were recorded for 2/5 male rats on both Day 8 and 15, while all other rats achieved satisfactory bodyweight gains throughout the study. No macroscopic abnormalities were observed during the macroscopic evaluation. Based on the results of the conducted study, a LD50 value of > 2000 mg/kg bw was derived for male and female rats.

Acute dermal toxicity

In an acute dermal toxicity study conducted under GLP conditions and according to OECD guideline 402, five male and female Wistar rats were exposed for 24 h to 2,5-bis(1,1,3,3-tetramethylbutyl)hydroquinone (CAS 903-19-5) in propylene glycol at a dose level of 2000 mg/kg bw under occlusive conditions (Key, 1998). The fur of all rats was clipped one day prior to testing. The animals were observed for 14 days for signs of systemic toxicity and mortality. No mortality occurred during the study period. Red staining of the head, neck and / or periorbital region was noted in 2/5 males and 1/5 females on Day 2. Erythema (Grade 1), scales and scabs were seen in the treated skin-area among 2/5 males and 3/5 females between Days 3 and 6. Brown staining of the skin on the back by the test substance was noted in 2/5 females on Day 2. The changes noted in mean body weight gain in males and females were within the range expected for rats used in this type of study and were therefore considered not indicative of toxicity.Pelvic dilatation in the right kidney, found in one female at macroscopic post mortem examination, is commonly noted among rats of this age and strain and was therefore not considered to be toxicologically significant. No further abnormalities were noted among the animals. Based on the results of the conducted study, the LD50 value was derived to be > 2000 mg/kg bw for male and female rats.

Justification for classification or non-classification

The available data on acute oral and dermal toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.

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