Manganese

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Not to GLP, follows basic scientific principles. Level of manganese ingested by the two exposed groups varied considerably within the dosing groups.

Remarks:

Study conducted on read-across material

Justification for type of information:

This study has been used to address the reproductive toxicity data requirements of manganese metal on the basis that a negative result for a soluble inorganic manganese compound is a worst case and therefore should demonstrate the lack of any developmental toxicity potential if compared to the bioavailable concentration of Mn.

Cross-reference

Reason / purpose for cross-reference:

other: Read-across target

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Justification for type of information:

This study has been used to address the reproductive toxicity data requirements of manganese metal on the basis that a negative result for a soluble inorganic manganese compound is a worst case and therefore should demonstrate the lack of any developmental toxicity potential if compared to the bioavailable concentration of Mn.

Reason / purpose for cross-reference:

read-across source

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

>= 325 - <= 678 other: mg/kg

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Air righting reflex delayed (Developmental parameter) in low protein group

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

>= 354 - <= 715 other: mg/kg

Based on:

not specified

Sex:

not specified

Basis for effect level:

other: Air righting reflex delayed (Developmental parameter) in normal protein group.

Reproductive effects observed:

not specified

Data source

Reference

Reference Type:

publication

Title:

Effect of low protein diet on manganese neurotoxicity: I. Developmental and biochemical changes

Author:

Ali MM, Murthy RC, Saxena DK, Srivastava RS and Chandra SV

Year:

1983

Bibliographic source:

Neurobehavioural toxicology and teratology, 5: 377-383

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The effects of a low protein diet (19% casein) and manganese exposure (Mn2+, 3 mg/mL drinking water) in rats was studied. The effect on growing (F0-90 days), rehabilitated (F0 low-normal protein- 28 days) and F1 generation pups was studied.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

managanese dichloride

IUPAC Name:

managanese dichloride

Details on test material:

- Name of test material (as cited in study report): Manganese chloride
- Analytical purity: Not reported

Test animals

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: ITRC colony bred
- Weight at study initiation: 40-45 g
- Housing: Acrylic cages


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±2°C
- Photoperiod (hrs dark / hrs light): 12 hr light:dark cycle

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

VEHICLE
- Choice of vehicle : Drinking water
- Concentration in vehicle: 3 mg/mL
- Amount of vehicle : Drinking water containing Mn was available ad libitum

DIET

Rats were divided randomly into two dietary groups: one group received a synthetic diet containing 21% casein and the other received a low protein diet containing 10% casein. Half of the rats in each dietary group were given drinking water containing MnCl2 at 3 mg/mL.
In addition some male rats on the low protein diet were given a normal protein diet for 28 days, the Mn exposure schedule remained the same. At the end of 28-day rehabilitation animals were sacrificed.

Details on mating procedure:

- M/F ratio per cage: 75 male to 50 female rats
- After successful mating each pregnant female was caged. Rats were isolated and kept singly in plastic cages and allowed to deliver normally.

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Details on study schedule:

Not reported

Doses / concentrations

Remarks:

Doses / Concentrations:
3 mg/mL water
Basis:
nominal in water

No. of animals per sex per dose:

21% casein (group 1): 37 male and 27 female
10% casein (group 2: 37 male and 27 female

Control animals:

other: Yes, normal protein diet without Mn exposure

Details on study design:

Estimation of manganese consumption (for individual animals):
((Mn2+ concentration mg/mL x water consumed over 24 hours mL)/ Body weight g) x 1000

Positive control:

Not reported

Examinations

Parental animals: Observations and examinations:

Growth pattern, diet consumption, water consumption, brain weight, brain Mn content, body weight assessed.

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

Not reported

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter redistributed within the groups to dams who had delivered on the same day.


PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: litter size, eye opening, startle index, air rightening index, viability index and lactation index.


GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.]

Postmortem examinations (parental animals):

ORGAN WEIGHTS
The effect of the protein diet and manganese administration on brain weight was examined.

Postmortem examinations (offspring):

ORGAN WEIGHTS
The effect of the protein diet and manganese administration on brain weight was examined.

Statistics:

All results, excluding developmental changes, were analysed by Student's t-test. For the developmental changes, a one way analysis of variance was applied to determine the significance of the difference of the means between the groups. The technique was applied after ascertaining the homogenicity of variance and normality assumptions of the data. The effect of Mn2+ in the low and normal protein fed rats were compared with their respective controls and in addition, the effect of low protein diet was assessed by comparing the low and normal protein fed control groups. Differences at p < 0.05 were considered significant.

Reproductive indices:

Not reported

Offspring viability indices:

Not reported

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Rats maintained on a low protein diet had a marked growth retardation compared to those on the normal protein diet. Mn2+ exposure in either dietary groups had no significant effect on the body weight growth pattern. No effects were noted on dietary consumption. The animals in the dietary rehabilitation group gained weight rapidly over the 28 days of rehabilitation (31-35%). The normal protein fed rats had a weight gain of only 8-10% during this period.


TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Water consumption in controls maintained on either diet was not found to differ (range 30-60 mL/day/rat over the entire experimental period). In Mn2+ exposed groups the diet exerted no effect on water consumption, however the volume of water consumed was much less compared to the controls (6-42 mL/day/rat). In the rehabilitated groups (control and Mn2+ exposed groups was less (18-34 mL/rat/day) compared to control groups (40-60 mL/rat/day).


ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects were noted on the brain weights in any treatment groups or the rehabilitation group.


BIOCHEMICAL PARAMETERS (PARENTAL ANIMALS): (Brain and plasma protein and brain Mn2+ content).
No effects were noted in the brain protein level in either diet or dosing group, this was also true for the rehabilitation group. However plasma levels were found to be reduced in the lower protein diet regimen. No effects were found to correlate with Mn2+ exposure. Plasma protein levels were found to be increased in rehabilitated groups, in the control as well as the Mn2+ groups, over the rehabilitation levels, recovering to those fed the normal protein diet. Mn+2 was again found to have no effect. Administration of manganese was found to increase the Mn2+ content in the brain in every dosing group, including the rehabilitated group.

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

LITTER SIZE (OFFSPRING)
The litter size of the pups born to the low protein fed dams was significantly lower than those born to the normal protein fed dams. Mn2+ exposure, in either dietary groups was found to have no significant effect on the litter size.


CLINICAL SIGNS (OFFSPRING): Developmental indices.
a). Eye opening: The age at opening both eyes was found to be significantly delayed in the pups born to protein deficient dams. Exposure to Mn2+ was found to have no significant effect. b). Auditory startle reflex: The age at which the pups first exhibited the auditory startle reflex was significantly delayed in the pups from protein deficient mothers. Mn2+ exposure in the low protein groups further delayed the appearance of this response, however it had no significant effect in the pups born to normal protein fed mothers dosed with Mn2+. c). Air righting: The age at which air righting reflex was noted was significantly delayed in the pups born to protein fed mothers. Mn2+ exposure caused further delay in the protein deficient groups. d). Viability and lactation indices: No significant effect on the viability and lactation indices in the pups of either dietary group either due to the dietary or Mn2+ exposure schedules.


TEST SUBSTANCE INTAKE (DAMS)
The dietary and Mn2+ exposure had no effect on food consumption rate of the dams during gestation and lactation. The water intake in the control and Mn2+ exposed groups on both dietary schedules was found to be slightly increased during the lactation period (control 47-75 mL and Mn2+ exposed 28-63 mL/day/rat) than during the gestation period (control 35-56 mL and experimental 20-52 mL/day/rat).


BODY WEIGHT (OFFSPRING)
The body weights of the low protein fed dams, both control and Mn2+ exposed were retarded by 37-45 % throughout the gestation and lactation periods, compared to the normal protein fed groups. F1 pups' body weights born to low protein dams were found to be retarded by 26, 16, 37, 46 and 43 % on days 0, 4, 7, 14 and 21 days of age respectively compared to those born to the normal protein fed dams.


ORGAN WEIGHTS (OFFSPRING)
The brain weight of the pups born from the low protein fed dams was found to be significantly lower than that of the offspring of the normal protein fed ones. Mn2+ exposure had no significant effect in either dietary group.


BIOCHEMICAL PARAMETERS (OFFSPRING): (Brain and plasma protein and brain Mn2+ content).
The brain protein level of the pups from the protein deficient dams was found to be considerably lower than pups to mothers fed a normal protein diet. The plasma protein levels of pups from protein deficient dams was decreased significantly and the Mn2+ exposure schedules in either dietary group were found to have no significant effect on the brain and plasma protein levels. The brain Mn2+ contents in the pups of either dietary group was found to be considerably higher than that found in the adults, the extent of the accumulation was the same in both dietary groups.

Effect levels (F1)

1

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

>= 325 - <= 678 other: mg/kg

Sex:

not specified

Basis for effect level:

other: Air righting reflex delayed (Developmental parameter) in low protein group

2

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

>= 354 - <= 715 other: mg/kg

Sex:

not specified

Basis for effect level:

other: Air righting reflex delayed (Developmental parameter) in normal protein group.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Effect of Concurrent Low Protein Diet and Mn2+ Exposure (90) Days in F0-Growing Rats

 

Group	Body Weight		Brain weight g	Brain weight/ Body weight	Protein contents			Brain Mn^2+content
	g	% Change			Brain mg/g	Plasma g/100 mL	% Change	µg/g	% Change
Normal protein	308.0 ± 28		1.59 ± 0.06	0.0051 ± 0.0002	147.0 ± 2.0	8.6 ± 0.11		0.84 ± 0.15
§Normal protein + Mn^2+	298.0 ± 32	N.S.*	1.61 ± 0.14	0.0054 ± 0.0004	150.0 ± 3.1	8.8 ± 0.34	N.S.*	1.84 ± 0.13	†120* p<0.001
Low protein	164.0 ± 19.5	-47† p<0.001	1.58 ± 0.08	0.0096 ± 0.0006	140.0 ± 2.9	6.2 ± 0.10	-28† p<0.001	0.86 ± 0.10	N.S.†
¶Low protein + Mn^2+	158.0 ± 14	N.S.‡	1.58 ± 0.12	0.01 ± 0.005	134.0 ± 9.4	6.0 ± 0.08	N.S.‡	1.86 ± 0.14	+116 p<0.001
Values represent mean ± S.E. of 6 rats; p values evaluated by Student’s t-test *† Compared to normal protein controls; ‡ Compared to low protein controls N.S. = Not significant Daily Mn^2+intake, mg/kg:       0-30 days  ¶377-473 §301-410                                                30-60 days ¶298-557 §240-495                                                60-90 days ¶305-675 §260-585

 

Table 2: Effect of Diet Rehabilitation (28 days) in F0-Rats Exposed to Concurrent Low Protein Diet and Mn²⁺ Exposure (90 days)

 

Group	Body Weight		Brain weight g	Brain weight/ Body weight	Protein contents			Brain Mn^2 +content
	g	% Change			Brain mg/g	Plasma g/100 mL	% Change	µg/g	% Change
Normal protein	338.0 ± 31		1.60 ± 1.10	0.0047 ± 0.0001	125.29 ± 2.99	8.39 ± 0.20		0.82 ± 0.09
§Normal protein + Mn^2+	333.0 ± 31	N.S.*	1.61 ± 0.09	0.0048 ± 0.0003	127.86 ± 2.96	8.22 ± 0.32	N.S.*	2.03 ± 0.08	†1.48* p<0.001
Low protein	266.0 ± 22	-21.3† p<0.05	1.60 ± 0.09	0.0060 ± 0.002	115.36 ± 4.17	8.22 ± 0.08	N.S.†	0.80 ± 0.06	N.S.†
¶Low protein + Mn^2+	250.0 ± 21	N.S.‡	1.58 ± 0.07	0.0063 ± 0.0003	119.78 ± 4.47	8.48 ± 0.32	N.S.‡	2.03 ± 0.05	+155‡ p<0.001
Values represent mean ± S.E. of 6 rats; p values evaluated by Student’s t-test *† Compared to normal protein controls; ‡ Compared to low protein controls N.S. = Not significant Daily Mn^2+intake, mg/kg:       ¶725-655                                                §315-610

 

Table 3: Effect of Concurrent Low Protein Diet and Mn2+ Exposure on the Appearance of Certain Developmental Landmarks and Indices in F1-Pups

 

Group	Litter Size	Day of			Viability index	Lactation index
		Eye opening	Auditory startle reflex	Air righting reflex
Normal protein	10.31 ± 0.73	15.31 ± 0.65	12.37 ± 0.79	16.25 ± 0.97	99.26 ± 6.2	95.22 ± 5.6
§Normal protein + Mn^2+	9.25 ± 0.55 N.S.*	15.58 ± 0.93 N.S.*	13.10 ± 1.10 N.S.*	19.74 ± 0.93 p<0.001*	88.24 ± 4.7 N.S.*	91.68 ± 8.2 N.S.*
Low protein	8.13 ± 0.15 p<0.01†	18.27 ± 0.85 p<0.001†	15.96 ± 0.92 p<0.001†	20.14 ± 1.21 p<0.001†	95.64 ± 5.2 N.S.†	90.40 ± 7.2 N.S.†
¶Low protein + Mn^2+	7.65 ± 0.64 N.S.‡	17.9 ± 1.2 N.S.‡	25.61 ± 1.17 p<0.01‡	25.61 ± 1.17 p<0.001‡	82.21 ± 4.3 N.S.‡	89.76 ± 6.5 N.S.‡
Values represent mean ± S.E. of 10-14 pups (litter mate); Statistical significance evaluated by ANOVA *† Compared to normal protein controls; ‡ Compared to low protein controls N.S. = Not significant Daily Mn^2+intake, mg/kg:       ¶325-678                                                §354-715

 

Table 4: Effect of Concurrent Low Protein Diet and Mn2+ Exposure in F1-Pups (Weaned)

Group	Body weight		Brain weight		Brain weight/ Body weight	Protein contents				Brain Mn^2+content
	g	% Change	g	% Change		Brain mg/g	% Change	Plasma g/100 mL	% Change	µg/g	% Change
Normal protein	43.7 ± 2.3		1.40 ± 0.09		0.0320 ± 0.0009	120.0 ± 3.0		8.4 ± 0.2		0.87 ± 0.14
§Normal protein + Mn^2+	41.4 ± 2.2	N.S.*	1.38 ± 1.03	N.S.*	0.0333 ± 0.0006	115.0 ± 4.0	N.S.*	8.7 ± 0.4	N.S.*	2.45 ± 0.10	+180* p<0.001
Low protein	24.8 ± 1.7	-43.2† P<0.001	1.13 ± 0.05	119† P<0.05	0.0455 ± 0.002	105.4 ± 3.0	-12† p<0.05	5.5 ± 0.2	-35† p<0.001	0.82 ± 0.09	N.S.†
¶Low protein + Mn^2+	23.6 ± 1.9	N.S.‡	1.15 ± 0.07	N.S.‡	0.0487 ± 0.0007	110.5 ± 1.63	N.S.‡	5.9 ± 0.2	N.S.‡	2.28 ± 0.08	+177‡ p<0.001
Values represent mean ± S.E. of 6 pups; Statistical significance evaluated by Student’s t-test *† Compared to normal protein controls; ‡ Compared to low protein controls N.S. = Not significant Daily Mn^2+intake of dams during gestation and lactation, mg/kg:          ¶325-678                                                                                                           §354-715

Applicant's summary and conclusion

Conclusions:

Mn exposure had no significant effect on growth pattern, brain weight or brain and plasma protein contents in either dietary group. Diet regimen had no effect on accumulation of Mn in any group but levels were higher in F1 pups. In F1 pups Mn exposure had no effect on eye opening in either group, delayed startle reflex in low protein group only but air righting reflex development delayed in both dietary groups, more marked in low protein group.