RSS-4, DRY SUBSTANCE

Endpoint summary

Description of key information

In an acute oral toxicity study as well as in an acute dermal toxicity study, the LD50 of the substance was determined to be >2000 mg/kg.

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:

2008/11/11-2008/12/10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted 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

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test type:

fixed dose procedure

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
Harlan Laboratories UK Ltd., Bicester, Oxon, Uk.
- Age at study initiation:
8-12 weeks.
- Weight at study initiation:
156-173g
- Fasting period before study:
Overnight
- Housing:
suspended solid-floor polypropylene cages furnished with wood flakes.
- Diet (e.g. ad libitum):
Ad libitum, apart from overnight period of fasting before initiation of study and approximately 3-4 hours after dosing.
- Water (e.g. ad libitum):
Ad libitum, apart from overnight period of fasting before initiation of study and approximately 3-4 hours after dosing.
- Acclimation period:
At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):
19-25 deg C
- Humidity (%):
70%
- Air changes (per hr):
15 changes per hour
- Photoperiod (hrs dark / hrs light):
12 hours dark/12 hours light


IN-LIFE DATES: From: Day 1 To: Day 14

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle:
300mg/kg dose level: 30 mg/ml
2000mg/kg dose test: 200 mg/ml
- Amount of vehicle (if gavage):
300mg/kg dose level: 10 mg/kg
2000mg/kg dose test: 10 mg/kg
- Justification for choice of vehicle:
Arachia Oil BP was used because the test material did not dissolve/suspend in distilled water.
- Lot/batch no. (if required):
CTO-35
- Purity:
Not stated.


MAXIMUM DOSE VOLUME APPLIED:
200mg/kg

DOSAGE PREPARATION (if unusual):
Not applicable.

CLASS METHOD (if applicable)
- Rationale for the selection of the starting dose:
In the absence of data regarding the toxicity of the test material, 300 mg/kg was chosen as the starting dose.

Doses:

300mg/kg
2000mg/kg

No. of animals per sex per dose:

300mg/kg dose: 1 female
2000mg/kg dose: 5 female

Control animals:

no

Details on study design:

- Duration of observation period following administration:
14 days
- Frequency of observations and weighing:
daily
- Necropsy of survivors performed:
Yes
- Other examinations performed:
clinical signs, body weight, organ weights, histopathology, behavoural abservations.

Statistics:

Not applicable.

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

-300 mg/kg bw group:
There was no mortality.

-2000 mg/kg bw group:
One animal treated was killed in extremis one day after dosing.

Clinical signs:

other: -300 mg/kg bw group: Signs of systemic toxicity noted during the day of dosing were hunched posture and pilo erection. The animal appeared normal four hours after dosing. -2000 mg/kg bw group: Signs of systemic toxicity noted were hunched posture, pilo-e

Gross pathology:

-300mg/kg bw group:
Necropsy findings are given in Table 3.
No abnormalities were noted at necropsy.

-2000 mg/kg bw group:
Individual necropsy findings are given in Table 6.
Abnormalities noted at necropsy of the animal that was killed in extremis were abnormally red lungs, dark liver and dark kidneys. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Other findings:

- Organ weights:
No abnormalities deteched.
- Histopathology:
Not stated.
- Potential target organs:
Not stated.
- Other observations:
Not stated.

Table1              Individual Clinical Observations and Mortality Data -300mg/kg

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
300	1-0 Female	HP	HP	HP	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

0= No signs of systemic toxicity

H = Hunched posture

P = Pilo-erection

Table2              Individual Bodyweights and Bodyweight Changes -300mg/kg

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight Gain (g) During Week
		0	7	14	1	2
300	1-0 Female	170	188	198	18	10

Table3              Necropsy Findings -300 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
300	1-0 Female	Killed Day 14	No abnormalities detected

Table4              Individual Clinical Observations and Mortality Data -2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Days)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	2-0 Female	0	0	HLARl	HLARlWt	HARl	H	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	H	HLA	HLAP	HLAP	HLAPRd	HPRd	H	H	0	0	0	0	0	0	0	0	0	0
	3-1 Female	H	HLA	HLAP	HLAP	HLAPRd (RrHoDh)X*
	3-2 Female	H	HLA	HLAP	HLAP	HP	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-3 Female	H	HLA	HLAP	HLAP	H	0	0	0	0	0	0	0	0	0	0	0	0	0

0= No signs of systemic toxicity

H = Hunched posture

P = Pilo-erection

L = Lethargy

A = Ataxia

Rd = Decreased respiratory rate

Rl = Laboured respiration

Wt = Tiptoe gait

Rr = Loss of righting reflex

Ho = Hypothermia

Dh = Dehydration

( ) = Observations noted prior to killingin extremis

X* = Animal killedin extremis

Table5              Individual Bodyweights and Bodyweight Changes-2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight (g) at Death	Bodyweight Gain (g) During Week
		0	7	14		1	2
2000	2-0 Female	168	170	184		2	14
	3-0 Female	160	167	179		7	12
	3-1 Female	173	-	-	162	-	-
	3-2 Female	159	176	180		17	4
	3-3 Female	156	166	183		10	17

Table6              Individual Necropsy Findings-2000mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2000	2-0 Female	Killed Day 14	No abnormalities detected
	3-0 Female	Killed Day 14	No abnormalities detected
	3-1 Female	Killedinextremis Day 1	Lungs: abnormally red Liver: dark Kidneys: dark
	3-2 Female	Killed Day 14	No abnormalities detected
	3-3 Female	Killed Day 14	No abnormalities detected

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 material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight (Globally Harmonised Classification System - Category 5).

Executive summary:

Introduction. 

The study was performed to assess the acute oral toxicity of the test material in the Wistar strain rat. The method was designed to meet the requirements of the following:

§        OECD Guidelines for Testing of Chemicals No 420 “Acute Oral Toxicity - Fixed Dose Method” (2001)

§        Method B1bisAcute Toxicity (Oral) of Commission Directive 2004/73/EC

Method. 

Following a sighting test at dose levels of300 mg/kg and2000 mg/kg, a further group of four fasted females was given a single oral dose of test material, as asuspensioninarachis oil BP, at a dose level of2000 mg/kg bodyweight. Clinical signs and bodyweight development were monitored during the study. All animals were subjected to gross necropsy.

Mortality.

One animal treated at a dose level of 2000 mg/kg was killedin extremisone day after dosing. There were no deaths noted at a dose level of 300 mg/kg.

Clinical Observations.

Signs of systemic toxicity noted in both dose groups were hunched posture and pilo-erection. Additional signs of systemic toxicity noted in animals treated at a dose level of 2000 mg/kg were lethargy, ataxia, decreased respiratory rate, laboured respiration, tiptoe gait, loss of righting reflex, hypothermia and dehydration. Surviving animals appeared normal four hours or two to five days after dosing.

Bodyweight.

Surviving animals showed expected gains in bodyweight.

Necropsy. 

Abnormalities noted at necropsy of the animal that was killedin extremiswere abnormally red lungs, dark liver and dark kidneys. No abnormalities were noted at necropsy of animals that were killed at the end of the study.

Conclusion. The acute oral median lethal dose (LD₅₀) of the test material in the female Wistar strain rat was estimated to be greater than 2000 mg/kg bodyweight (Globally Harmonised Classification System-Category 5).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The study has Klimisch score 1

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:

14 - 28 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000; including the most recent partial revisions.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: Wistar strain, Crl:WI (Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: Young adult animals (approx. 12 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 301-376 grams; females: 211-242 grams)
- Housing: Individual housing in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES: From: 14 - 28 March 2012

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

One day before exposure (Day -1) an area of approximately 5x7 cm on the back of the animal was clipped.

The formulation was applied in an area of approx. 10% of the total body surface, i.e. approx. 25 cm² for males and 18 cm² for females. The formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy 1D)*, successively covered with aluminum foil and Coban elastic bandage*. A piece of Micropore tape* was additionally used for fixation of the bandages in females only. *. Manufacturers: Laboratoires Stella s.a., Liege, Belgium (surgical gauze) and 3M, St. Paul, Minnesota, U.S.A. (Coban & Micropore).

Frequency: Single dosage, on Day 1.

Washing: Following application, dressings were removed and the skin cleaned of residual test substance using tap water.

Duration of exposure:

24 hours.

Doses:

2000 mg/kg

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

VEHICLE
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Dose volume: 10 mL/kg

DOSAGE PREPARATION: The formulation (w/w) was prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level. No correction was made for purity of the test substance.

Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
Mortality/Viability: Twice daily.
Body weights: Days 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: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.
- Other examinations performed: none.

Statistics:

None.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Hunched posture was shown by two females on Day 2. Chromodacryorrhoea was shown by all animals on Day 1. Scabs and/or scales were noted on the treated skin-area of one male and two females during the observation period.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Other findings:

None

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on these results, Magnesium bis (2-ethylbutanoate) does not have to be classified and has no obligatory labelling requirement for acute dermal toxicity according to the:
- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011),
- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The study has Klimisch score 1

Additional information

Acute toxicity: oral

An acute oral toxicity study was performed with the substance according to OECD 420 (2001) and GLP principles. Dosing started with 300 mg/kg (1 female) and 2000 mg/kg (1 female), with addition of another 4 females dosed at 2000 mg/kg bodyweight. 

One rat dosed 2000 mg/kg was killed in extremis on Day 1, after dosing. Clinical signs noted at both dose levels included hunched posture and pilo-erection. At 2000 mg/kg lethargy, ataxia, decreased respiratory rate/laboured respiration, tiptoe gait, loss of righting reflex, hypothermia and dehydration were observed. Animals appeared normal 2-5 days after dosing. No abnormalities were observed at macroscopic examination. Based on these results, the acute oral LD50 of the substance was determined to be >2000 mg/kg.

Acute toxicity: dermal

An acute dermal toxicity study was performed with the substance according to OECD 402 (1987) and GLP principles. A total of 5 rats per sex were dermally exposed to a dose of 2000 mg/kg for 24 hours under occlusive conditions. No mortality occurred. Hunched posture was shown by two females on Day 2. Chromodacryorrhoea was shown by all animals on Day 1. Scabs and/or scales were noted on the treated skin-area of one male and two females during the observation period. No abnormalities were found on bodyweight gain and at macroscopic post mortem examination of the animals. Based on these results, the acute dermal LD50 of the substance was determined to be >2000 mg/kg.

Justification for selection of acute toxicity – oral endpoint
Only one study available

Justification for selection of acute toxicity – dermal endpoint
Only one study available

Justification for classification or non-classification

As the acute oral LD50 and the acute dermal LD50 were determined to be >2000 mg/kg, the substance does not need to be classified for acute oral and acute dermal toxicity in accordance with the CLP Regulation.