Trimanganese bis(orthophosphate)

Endpoint summary

Description of key information

Acute oral toxicity: LD50 (female rats) > 2000 mg/kg bw (OECD 420; GLP)

Acute inhalation toxicity: LC50 (male and female rats; 4 hours) > 5.07 mg/L (actual concentration) (OECD 436; GLP)

Acute dermal toxicity: data waiving

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:

2012-11-06 to 2012-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2001-12-17

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2012-11-30

Test type:

fixed dose procedure

Limit test:

no

Specific details on test material used for the study:

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

Species:

rat

Strain:

Wistar

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: 151-167 g
- Fasting period before study: overnight
- Housing: animals were housed in groups of up to four in suspended solid-floor polypropylene cages furnished with woodflakes.
- Diet: 2014C Teklad Global Rodent diet (Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

arachis oil

Remarks:

BP

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 30 and 200 mg/mL
- Justification for choice of vehicle: Arachis oil BP was used because the test item was considered more suitable for dosing in this vehicle.

MAXIMUM DOSE VOLUME APPLIED: 10 mL/kg bw

- Rationale for the selection of the starting dose: In the absence of data regarding the toxicity of the test item, 300 mg/kg was chosen as the starting dose.

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

300 mg/kg bw: 1
2000 mg/kg: 5

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 fourteen days. Morbidity and mortality checks were made twice daily. Individual body weights were recorded on Day 0 (the day of dosing) and on Days 7 and 14.
- Necropsy of survivors performed: yes

Statistics:

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

Preliminary study:

There was no mortality. No signs of systemic toxicity were noted during the observation period (Table 1).
The animal showed expected gains in bodyweight over the observation period (Table 2).
No abnormalities were noted at necropsy (Table 3).

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

There were no deaths.
Please refer to table 4 in the field "Any other information on results incl. tables" below.

Clinical signs:

other: No signs of systemic toxicity were noted during the observation period. Please refer to table 4 in the field "Any other information on results incl. tables" below.

Gross pathology:

No abnormalities were noted at necropsy.
Please refer to table 6 in the field "Any other information on results incl. tables" below.

Table 1: Individual Clinical Observations and Mortality Data – 300 mg/kg

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

Table 2: Individual Bodyweights and Bodyweight Changes – 300 mg/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	166	198	222	32	24

 

Table 3: 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

Table 4: Individual Clinical Observations and Mortality Data – 2000 mg/kg

Dose Level (mg/kg)	Animal Number and Sex	Effects Noted After Dosing (Hours)				Effects Noted During Period After Dosing (Day)
		½	1	2	4	1	2	3	4	5	6	7	8	9	10	11	12	13	14
2000	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

Table 5: Individual Bodyweights and Bodyweight Changes – 2000 mg/kg

Dose Level mg/kg	Animal Number and Sex	Bodyweight (g) at Day			Bodyweight Gain (g) During Week
		0	7	14	1	2
2000	2-0 Female	155	176	191	21	15
	3-0 Female	156	181	200	25	19
	3-1 Female	152	173	195	21	22
	3-2 Female	167	194	215	27	21
	3-3 Female	151	174	186	23	12

Table 6: Necropsy Findings – 2000 mg/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	Killed Day 14	No abnormalities detected
	3-2 Female	Killed Day 14	No abnormalities detected
	3-3 Female	Killed Day 14	No abnormalities detected

Interpretation of results:

GHS criteria not met

Conclusions:

LD50 (female rats) > 2000 mg/kg bw
According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not acutely toxic via the oral route.

Executive summary:

Introduction

The study was performed to assess the acute oral toxicity of the test item in the Wistar strain rat. The method was designed to be compatible with the following:

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

 Method B1 bis Acute Toxicity (Oral) of Commission Regulation (EC) No. 440/2008

Method

Following a sighting test using one animal at each dose level of 300 mg/kg and 2000 mg/kg, a further group of four fasted females was given a single oral dose of test item, as a suspension in arachis oil BP, at a dose level of 2000 mg/kg body weight. Clinical signs and body weight development were monitored over a 14-day observation period. All animals were subjected to gross necropsy.

Mortality.There were no deaths.

Clinical Observations.There were no signs of systemic toxicity.

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

Necropsy. No abnormalities were noted at necropsy.

Conclusion.The acute oral median lethal dose (LD50) of the test item in the female Wistar strain rat was estimated to be greater than 2000 mg/kg body weight. The test item does not meet the criteria for classification according to the Globally Harmonised Classification System or Regulation (EC) No 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-04-01 to 2015-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 436 (Acute Inhalation Toxicity: Acute Toxic Class Method)

Version / remarks:

2009-09-07

Deviations:

yes

Remarks:

GSD on particle size determinations were > 3 µm; rel. humidity was < 30% during exposure

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2013-11-27

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature, protected from light

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item was desiccated 18 hours to facilitate the generation of a respirable aerosol.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Barcelona, Spain
- Age at study initiation: 8 weeks
- Weight at study initiation: males: 260.06-279.34 g; females: 200.83-214.44 g
- Housing: 4 animals per cage before distribution, 3 animals per cage after distribution; bedding material: Capsumlab Lecho_10 (autoclavable)
- Diet: Global Diet 2914 C (Harlan Teklad, UK), ad libitum, except when animals were restrained in the exposure tubes
- Water: Tap water, ad libitum, except when animals were restrained in the exposure tubes
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature: 16.9-23.7 °C
- Relative humidity: 21 - 52 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

1.4 µm

Remark on MMAD/GSD:

Mean Mass Median Aerodynamic Diameter (MMAD) of particle size distribution during exposure was calculated from two gravimetric measurements PSD #1 and PSD #2. Mean MMAD during exposure was 1.40 μm. This value is within the respirable range (1-4 μm) and appropriate for acute inhalation toxicity testing. Geometric Standard Deviation (GSD) on PSD #1 and PSD#2 were above the upper limit of 3 but considered acceptable as more than 63% of particles were below the upper limit of 4 μm in both cases (63.27% for PSD #1 and 66.7% for PSD#2) (see Table 1).

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Exposure chambers type EC-FPC-232 (anodised aluminium), equipped with glass exposure tubes were used.
- Exposure chamber volume: approximately 3 L
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
- Source and rate of air: Filtered air from a compressor. The exposure airflow rate was adjusted as appropriate before the start of the exposure using the pressure difference over a Venturi tube. The actual airflow rate was monitored hourly in each group during each exposure. The target range was 0.5-1.0 L/min through each inhalation tube.
- System of generating particulates/aerosols: A dust aerosol was generated from the desiccated test item using a Rotating Brush Generator PALAS RBG 2000. The dust was diluted with filtered air from a compressor and conveyed via glass tubing, from the generator to the exposure chamber. The flow rate through the exposure chamber was adjusted as necessary.
- Method of particle size determination: The particle size distribution was determined gravimetrically twice during exposure. The cumulative particle size distribution of the test aerosol was determined using a PIXE cascade impactor. The particle size distribution of the test item in the generated aerosol was measured by gravimetry analyzing the test item deposited on each stage of the cascade impactor.
The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel® software (Microsoft Corporation, USA). The target ranges were 1 to 4 μm for the MMAD and 1.5 to 3 for the GSD.
- Temperature, humidity in air chamber: The temperature in the chamber was measured continuously during exposure using a thermohygrometer (Kimoth110, Kimo). The target range was 19-25°C. The results were reported approximately hourly from the start of the inhalation exposure.
The relative humidity in the chamber was measured continuously during exposure using a thermohygrometer (Kimoth110, Kimo). The target range was 30-70%. The results were reported approximately hourly from the start of the inhalation exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: The test item usage was determined once per exposure by weighing the amount of the test item before and after exposure to determine the quantity of test item used. The weight used was then divided by the total air-flow volume to give the nominal concentration. These data were used for the purpose of monitoring the performance of the generation system.
Gravimetric determination of the aerosol concentration was performed at least once during each hour of exposure. Test aerosol samples were collected onto a Whatman filter (grade F319-04) using a filter sampling device. The sampling flow was similar to the air flow rate per exposure port. The duration of sampling was 5 minutes. The filters were weighed before and immediately after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: The starting dose (approximately 5 mg/L air, during 4 hours) was selected as no toxic effects were expected based on the available data. This concentration was determined during technical trials (see 'Any other information on materials and methods incl. tables').

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

5.07 mg/L ± 0.56 (actual concentration)
2.0 mg/L (target concentration)

No. of animals per sex per dose:

3 males / 3 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were examined daily for mortality and morbidity. Clinical observations in response to treatment were performed on all animals hourly during exposure (only grossly abnormal signs), immediately and 1h after exposure, and once daily thereafter until the end of the observation period. Any visible clinical signs and discomfort were recorded. All animals were weighed on the day of treatment, just before starting the inhalation period (Day 1 of study), daily from Days 2 to 11, and immediately before sacrifice on Day 15 of study.
- Necropsy of survivors performed: yes

Statistics:

No statistical analysis was performed.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.07 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals survived the scheduled observation period.

Clinical signs:

other: Dirty fur and chromorrhinorrhea were observed in all animals 1 hour after exposure. In addition, chromodacryorrhea (1 out of 3 males and in 1 out of 3 females) and mild piloerection (2 out of 3 males and in 2 out of 3 females) were recorded 1 hour after e

Body weight:

A significant body weight loss with respect to body weight at pretreatment (approximately 15% less in both sexes) was observed in all animals from day 1 of study to day 3 of study. Animals started to gain weight from day 4 of study but body weight at pretreatment was not recovered until day 10 of study in males and day 11 of study in females.
(See attached Appendix 2)

Gross pathology:

Lungs from 5 out of 6 animals did not show a normal appearance at the time of necropsy. The following macroscopic observations were recorded:
- White areas: 2 out of 6 animals (1 male and 1 female)
- Red spots: 2 out of 6 animals (1 male and 1 female)
- Black spots: 1 out of 6 animals (1 male)
- Pale appearance: 2 out of 6 animals (1 male and 1 female)
Lungs from affected animals were collected and preserved in fixative for further histopathological examination if considered necessary.
(See attached Appendix 3)

Test atmosphere conditions

Temperature during exposure was considered to be satisfactory and within target range (19-25ºC). Relative humidity was below target range (30-70%) (see section 13, deviation #3). Data are presented in the following table:

Recording time (h:min from exposure start)	Temperature (ºC)	Relative Humidity (%)
0:04	20.4	15.8
1:06	20.3	14.8
2:10	20.8	14.3
3:35	21.7	14.8
Mean	20.8	14.9
SD	0.64	0.63
N	4	4

Mean oxygen and carbon dioxide concentrations were 21.0 % and 0.04% respectively. These values are considered satisfactory for inhalation studies and within target range (at least 19% and below 1% respectively). Data are presented in the following table:

Recording time (h:min from exposure start)	Oxygen (%)	Carbon dioxide (%)
0:04	20.9	0.04
1:06	21.0	0.04
2:10	20.9	0.04
3:35	21.0	0.04
Mean	21.0	0.04
SD	0.06	0.00
N	4	4

 

Aerosol concentrations

The mean of the gravimetric concentrations during exposure was 5.07 mg/L air, as targeted. Data on aerosol concentrations are presented in the following table:

Group A (5.07 mg/L air). Day 1 of study
Sampling starting time (h:min from exposure start)	Sampling volume (L)	Amount of test item on the filter (mg)	Gravimetric aerosol concentration (mg/L)
0:06	5.54	26.47	4.78
0:36	5.57	31.27	5.62
1:01	5.57	31.85	5.72
1:12	5.57	29.57	5.31
1:45	5.57	30.70	5.52
2:11	5.56	26.44	4.76
2:31	4.43	19.45	4.39
2:40	5.56	24.69	4.44
3:08	5.56	24.67	4.44
3:39	5.56	26.88	4.84
3:49	5.56	32.81	5.91
MEAN	5.46	27.71	5.07
SD	0.34	3.98	0.56
N	11	11	11

 

Interpretation of results:

GHS criteria not met

Conclusions:

LC50 (male and female rats; 4 hours) > 5.07 mg/L(actual concentration)
According to the Regulation (EC) No 1272/2008 and subsequent adaptations, the substance is not acutely toxic via the inhalative route.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute oral toxicity

The acute oral toxicity of trimanganese bis(orthophosphate) was tested in female Wistar rats in a study following OECD Guideline 420 and under GLP conditions (Bradshaw, 2014). In the absence of data regarding the toxicity of the test item, one single animal was tested first at a starting dose of 300 mg/kg bw given by oral gavage. In the absence of mortality and any other signs of toxicity, a second animal was tested at an oral dose level of 2000 mg/kg bw. In the absence of toxic effects, a group of 4 animals were orally treated with the test material at 2000 mg/kg bw. There were no deaths and no signs of systemic toxicity were noted during the 14-day observation period. All animals showed expected gains in bodyweight. No abnormalities were noted at necropsy. The oral LD50 of the test material in female rats was thus determined to be > 2000 mg/kg bw.

Therefore, the test material does not fulfil the criteria for classification according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), and is thus considered to be not acutely toxic by the oral route.

Acute inhalation toxicity

The acute inhalation toxicity of trimanganese bis(orthophosphate) was evaluated in male and female Sprague Dawley rats by the acute toxic class (ATC) method according to OECD Guideline 436 and under GLP conditions (Veiga, 2015). A group of three male and three female rats was exposed by nose-only, flow-past inhalation to the test material at a mean concentration of 5.07 mg/L air during 4 hours. This concentration was found to be the highest technically achievable concentration. The mean Mass Median Aerodynamic Diameter (MMAD) during exposure was 1.4 µm. More than 63% of the particles were <4 µm. The test material was therefore considered to be respirable to rats.

Chromorrhinorrhea (6 out of 6 animals), dirty fur (6 out of 6 animals), chromodacryorrhea (2 out of 6 animals) and mild piloerection (4 out of 6 animals) were recorded 1 hour after exposure. Chromorrhinorrhea was not longer observed from day 3 of study onwards. Dirty fur was recorded up to day 3 in all males and up to day 4 of study in one female. On the other hand, mild to moderate piloerection was observed in both sexes up to day 6 of study. Lachrymation was only observed in 2 out of 3 females at day 3 of study. In addition, significant signs of toxicity such as apathy and decreased respiratory rate were recorded in all animals independently of sex at day 2, and in day 3 of study in 5 out of 6 animals. Finally, hunched posture was only observed in all females at day 3 and at day 4 of study (1 out of 3 females). No clinical signs were recorded from day of study 7 to the end of the observation period.

A significant body weight loss with respect to body weight at pretreatment (approximately 15% less in both sexes) was observed in all animals from day 1 of study to day 3 of study. Body weight at pretreatment was not recovered until day 10 of study in males and until day 11 of study in females.

Relevant macroscopic findings (white areas, red spots, black spots and pale appearance) were observed in the lungs of 5 out of 6 animals at the time of necropsy.

There was no indication of relevant sex-related differences in toxicity of test item.

The LC50 of the test material in male and female rats was determined to be >5.07 mg/L.

Therefore, the test material does not fulfil the criteria for classification and is thus considered to be not acutely toxic by the inhalation route according to Regulation (EC) No 1272/2008 (CLP). However, based on the observation of significant clinical signs of toxicity, significant body weight loss, and the relevant necropsy findings in the lungs, the test material is considered to fulfil the classification criteria for acute toxicity by inhalation Category 5 (Hazard phrase H333: May be harmful if inhaled) according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS).

Acute dermal toxicity

This information is not available.

Justification for classification or non-classification

Acute oral toxicity

The substance is not acutely toxic via the oral route based on an acute oral toxicity test (OECD 420) and does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.

Specific target organ toxicant (STOT) - single exposure: oral

Reversible or irreversible adverse health effects were not observed immediately or after exposure in an acute oral toxicity test (OECD 420).Thus, the classification criteria as specific target organ toxicant (STOT) – single exposure, oral are not met and the substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.

Acute inhalation toxicity

The substance is not acutely toxic via the inhalation route based on an acute inhalation toxicity test (OECD 436) and does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.

Specific target organ toxicant (STOT) – single exposure: inhalation

Reversible or irreversible adverse health effects were not observed immediately or after exposure in an acute inhalation toxicity test (OECD 436).Thus, the classification criteria as specific target organ toxicant (STOT) – single exposure, inhalation are not met and the substance does not require classification according to Regulation (EC) No 1272/2008 and subsequent adaptations.