Manganese dinitrate

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-04-07 to 2010-05-11.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP and to current guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

fixed dose procedure

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Manganese dinitrate tetrahydrate

IUPAC Name:

Manganese dinitrate tetrahydrate

Details on test material:

Sponsor's identification: Mn(NO3)2 • 4H2O
Description : pink solid
Batch number : not supplied
Date received : 19 November 2009
Expiry date : not supplied
Storage conditions: approximately 4°C in the dark over silica gel

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Female Wistar (HsdRccHan:WIST) strain rats were supplied by Harlan Laboratories UK Limited, Bicester, Oxon, UK.
- Age at study initiation: Eight to twelve weeks.
- Weight at study initiation: 153 - 189 g. The bodyweight variation did not exceed ± 20% of the initial/mean bodyweight of any previously dosed animal(s).
- 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 : Free access to food (2014 Teklad Global Rodent diet supplied by Harlan Teklad, Blackthorn, Bicester, Oxon, UK) was allowed throughout the study.
- Water : Free access to mains drinking water was allowed throughout the study.
- Acclimation period: At least five days

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 280.4 mg/ml and 42.1 mg/ml


MAXIMUM DOSE VOLUME APPLIED: 10 ml/kg

DOSAGE PREPARATION (if unusual): For the purpose of the study the test material was freshly prepared, as required, as a solution in distilled water.
The test material was formulated within two hours of being applied to the test system. It is assumed that the formulation was stable for this duration.


- Rationale for the selection of the starting dose: Using available information on the toxicity of the test material, 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) was chosen as the starting dose.

Doses:

Dose levels of 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) and 421 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight).

No. of animals per sex per dose:

A single female was treated at a dose level of 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight).
Five females were treated at a dose level of 421 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight).

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were made ½, 1, 2, and 4 hours after dosing and then daily for up to 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 or at death.
- Necropsy of survivors performed: yes.

Statistics:

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

Results and discussion

Preliminary study:

Dose Level - 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight):

Individual clinical observations and mortality data are given in Table 1.

Mortality:
The animal was killed for humane reasons two hours after dosing, due to the approach of the moderate severity limit set by the UK Home Office.

Clinical Observations:
Signs of systemic toxicity noted were hunched posture, lethargy, tiptoe gait, abdominal discomfort and pilo erection.

Bodyweight:
The bodyweight on Day 0 and at death are given in Table 2.

Necropsy:
Necropsy findings are given in Table 3.
Abnormalities noted at necropsy of the animal treated at a dose level of 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight) were patchy pallor of the liver, pale kidneys and severe haemorrhage of the gastric mucosa.

Based on the results at a dose level of 2804 mg/kg, a dose level of 421 mg/kg bodyweight was investigated.

Effect levels

Sex:

female

Dose descriptor:

LD50

Effect level:

> 300 mg/kg bw

Based on:

test mat.

Remarks:

anhydrous equivalent

Mortality:

Dose Level - 421 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight):
There were no deaths.
At 2804 mg/kg (equivalent to 2000 mg active ingredient/kg bodyweight, the single female rat tested had to be humanely killed.
Individual clinical observations and mortality data are given in Table 4.

Clinical signs:

other: Dose Level - 421 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight): No signs of systemic toxicity were noted during the observation period.

Gross pathology:

Dose Level - 421 mg/kg (equivalent to 300 mg active ingredient/kg bodyweight):
Individual necropsy findings are given in Table 6.
No abnormalities were noted at necropsy.

Any other information on results incl. tables

Table 1              Individual Clinical Observations and Mortality Data -2804 mg/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
2804+	1-0 Female	HLWt	HLWtAd	HLPWtX*

+ =     Equivalent to 2000 mg active ingredient/kg bodyweight)

H=      Hunched posture

L =      Lethargy

Wt =    Tiptoe gait

Ad =    Abdominal discomfort

P =      Pilo‑erection

X* =     Animal was killed for humane reasons due to the approach of the moderate severity limit set by the UK Home Office

Table 2              Individual Bodyweights and Bodyweight Changes -2804 mg/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
2804+	1-0 Female	153	-	-	151	-	-

+ =     Equivalent to 2000 mg active ingredient/kg bodyweight)

- = animal dead

Table 3              Necropsy Findings - 2804 mg/kg

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
2804+	1-0 Female	Humanely Killed Day 0	Liver: patchy pallor Kidneys: pale Gastric mucosa: severe haemorrhage

+ =     Equivalent to 2000 mg active ingredient/kg bodyweight)

Table 4              Individual Clinical Observations and Mortality Data - 421mg/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
421*	2-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-0 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-1 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-2 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	3-3 Female	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0

* =     Equivalent to 300 mg active ingredient/kg bodyweight)

0=     No signs of systemic toxicity

Table 5              Individual Bodyweights and Bodyweight Changes - 421mg/kg*

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight Gain (g) During Week
		0	7	14	1	2
421*	2-0 Female	171	184	193	10	9
	3-0 Female	156	160	168	4	8
	3-1 Female	184	189	200	5	11
	3-2 Female	162	167	177	5	10
	3-3 Female	189	193	209	4	16

* =     Equivalent to 300 mg active ingredient/kg bodyweight)

Table 6              Individual Necropsy Findings- 421mg/kg*

Dose Level mg/kg	Animal Number and Sex	Time of Death	Macroscopic Observations
421*	2-0 Female	Killed Day 14	No abnormalities detected
	3-0 Female	Killed Day 14	No abnormalities detected
	3-1 Female	Killed Day 14	No abnormalities detected
	3-2 Female	Killed Day 14	No abnormalities detected
	3-3 Female	Killed Day 14	No abnormalities detected

* =     Equivalent to 300 mg active ingredient/kg bodyweight)

Applicant's summary and conclusion

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The acute oral median lethal dose (LD50) of the test material in the female Wistar strain rat is greater than 300 mg/kg bodyweight (Globally Harmonised Classification System - Category 4). The effects seen in the single animal tested at the highest dose are most likely due to the severe local action of the test substance, rather than a true systemic effect.