Manganese di(acetate)

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across substance, guideline followed with devations, no raw data provided; however, the given data indicate that the study was well-performed and is sufficient to assess the effects of the test item on reproduction / fertility.

Data source

Reference

Reference Type:

publication

Title:

Effects of long-term exposure to manganese chloride on fertility of male and female mice

Author:

Elbetieha A, Bataineh H, Darmani H, Al-Hamood, MH

Year:

2001

Bibliographic source:

Toxicology Letters 119 (2001) 193-201

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Reproductive Part

Deviations:

yes

Remarks:

animals were sacrificed 10 days after a 10 day mating period, i.e. post-partum observation is missing, Gross pathology only performed partly, no dosing during mating period and pregnancy

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Manganese chloride

IUPAC Name:

Manganese chloride

Constituent 2

Reference substance name:

MnCl2*4H2O

IUPAC Name:

MnCl2*4H2O

Test material form:

not specified

Details on test material:

- Name of test material (as cited in study report): Manganese chloride
- Molecular formula (if other than submission substance): MnCl2*4H2O
- Supplier: Riedel-De-Haen AG, Seelze, Hannover, Germany

Test animals

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animal house unit in the Faculty of Medicine at Jordan University of Science and Technology
- Age at study initiation: (P) 50 days
- Weight at study initiation: (P) Males & Females: ≈28g
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): food (manufactured by the faculty of Veterinary Medicine at Jordan University of Science and Technology, Irbid, Jordan, according to standard recipies) had a manganese content of 60 mg/kg chow and was available ad libitum
- Water (e.g. ad libitum): available ad libitum, drinking water study, tap water itself contained 0.3 mg/l manganese
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 1 °C
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12:12 hours dark:light (Light from 6 AM to 6 PM)

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

unchanged (no vehicle)

Details on exposure:

Manganese chloride was dissolved in tap water.

Details on mating procedure:

EXPERIMENT 1
treated males / untreated females
- M/F ratio per cage: 1 male / 2 females
- Length of cohabitation: 10 days during which two estrous cycles should have elapsed
- Proof of pregnancy: no data
- After successful mating each pregnant female was caged (how): After ten days males were removed from females, two females were caged for 10 days
- Any other deviations from standard protocol: no dosing during mating / pregnancy


EXPERIMENT 2
untreated males / treated females
- M/F ratio per cage: 1 male / three females
- Length of cohabitation: 10 days during which two estrous cycles should have elapsed
- Proof of pregnancy: no data
- After successful mating each pregnant female was caged (how): After ten days males were removed from females, three females were caged for 10 days
- Any other deviations from standard protocol: no dosing during mating / pregnancy

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

12 weeks prior to mating

Frequency of treatment:

Test item was dissolved in drinking water which was constantly available ad libitum

Details on study schedule:

- Age at mating of the mated animals in the study: 19 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
1000, 2000, 4000, 8000 mg/l
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
108.3 ± 6.34, 172 ± 13.02, 352 ± 14.91, 706.5 ± 18.26 mg/kg bw/day
Basis:
other: actual ingested, calculated from daily water consumption of males

Doses / concentrations 3

Remarks:

Doses / Concentrations:
99.83 ± 8.61, 187.54 ± 9.34, 358.84 ± 14.16, 634.92 ± 21.52 mg/kg bw/day
Basis:
other: actual ingested, calculated from daily water consumption of females

No. of animals per sex per dose:

14 treated males, 28 untreated females (Experiment 1), 5 untreated males, 15 treated females (Experiment 2)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: doses were chosen according previous studies (Sanchez DJ et al. (1993) Toxicol. Lett. 69, 45-52)

Positive control:

no data

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: after sacrification

BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

Oestrous cyclicity (parental animals):

No data on monitoring; it was assumed that within the 10 day mating period two estrous cycles should have elapsed

Sperm parameters (parental animals):

no data

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 fetuses: number of implantations and viable fetuses

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: no data
- Maternal animals: All surviving animals ten days after mating period

HISTOPATHOLOGY / ORGAN WEIGHTS
Body, qvars and uterus were weighted

Postmortem examinations (offspring):

not applicable

Statistics:

Data were expressed as means ± SD (standard deviation). Differences between control and manganese chloride exposed groups were analyzed using Fisher´s exact test (two-tail) and Student´s t-test. The P values of <0.05 were considered statistically significant.

Reproductive indices:

no data

Offspring viability indices:

no data

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

body weights

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

body weights

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

ambigous regarding treated males / females

Details on results (P0)

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Only females were exermined and no substance-related or any other effects on body weight were observed

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Water consumption decreased relative to control by increasing MnCl2 dose which is most probably to the taste of the test substance; actual ingested dose was calculated regarding water consumption. No data on time-dependence of compound intake were given.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Regarding treated males, a dose dependent decrease of pregnant females was observed
Regarding treated females, no statistically significant effect on the number of pregnant females was observed.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Both relative weights of ovars and uterus increased dose-dependently

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

ca. 634.92 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks:

MnCl2

Sex:

female

Basis for effect level:

other: mortality; body weight; pregnancy index, number of resorptions (highest dose level tested)

2

Dose descriptor:

NOAEL

Effect level:

ca. 358.84 mg/kg bw/day

Based on:

test mat.

Remarks:

MnCl2

Sex:

female

Basis for effect level:

other: number of implantations; viability index (fetuses)

3

Dose descriptor:

NOAEL

Effect level:

ca. 187.54 mg/kg bw/day

Based on:

test mat.

Remarks:

MnCl2

Sex:

female

Basis for effect level:

other: relative ovarian weights

4

Dose descriptor:

LOAEL

Effect level:

ca. 99.83 mg/kg bw/day

Based on:

test mat.

Remarks:

MnCl2

Sex:

female

Basis for effect level:

other: relative uterine weight

5

Dose descriptor:

NOAEL

Effect level:

ca. 706.5 mg/kg bw/day

Based on:

test mat.

Remarks:

MnCl2

Sex:

male

Basis for effect level:

other: Number of implantations, number of viable fetuses, total number of resorptions (highest tested dose level)

6

Dose descriptor:

NOAEL

Effect level:

ca. 352 mg/kg bw/day

Based on:

test mat.

Remarks:

MnCl2

Sex:

male

Basis for effect level:

other: pregnancy index

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

viability of fetuses

Body weight and weight changes:

not specified

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
The number of viable fetuses decreased dose-dependently

Effect levels (F1)

Remarks on result:

other: no information available

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Effect of long-term exposure to manganese chloride via drinking water on fertility of male mice

MnCl2(mg/ml drinking water)	MnCl2 (mg/kg bw/day)^#	No. of males	No. of females	No. of pregnant females(%)	No. of implantations^#	No. of viable fetuses^#	Total No. of resorptions
Control	0	14	28	26 (92)	9.00 ± 2.22	8.76 ± 3.35	7
1000	108.3 ± 6.34	14	28	25 (89)	8.73 ± 1.68	8.50 ± 1.74	13
2000	172 ± 13.02	14	28	22 (78)	8.86 ± 1.75	8.40 ± 2.23	10
4000	352 ± 14.91	14	28	20 (71)	8.15 ± 1.81	7.60 ± 1.87	11
8000	706.5 ± 18.26	14	28	17 (66)*	8.00 ± 1.96	7.70 ± 1.89	6

^#results are expressed as mean ± SD

* P < 0.05, significantly different compared to control value (Fisher´s exact test, two-tail)

 

Table 2: Effect of long-term exposure to manganese chloride via drinking water on fertility of female mice

MnCl2(mg/ml drinking water)	MnCl2 (mg/kg bw/day)^#	No. of females	No. of pregnant females (%)	No. of implantations^#	No. of viable fetuses^#	No. of mice with resorptions (%)	Total No. of resorptions
Control	0	15	13 (86)	9.41 ± 1.68	9.41 ± 1.68	0/13 (0)	0
1000	99.83 ± 8.61	15	13 (86)	9.08 ± 1.62	9.00 ± 1.68	3/13 (23)	3
2000	187.54 ± 9.34	15	13 (86)	8.42 ± 1.92	8.25 ± 2.05	2/13 (15)	2
4000	358.84 ± 14.16	15	9 (60)	8.43 ± 2.38	8.28 ± 2.22	1/9 (11)	1
8000	634.92 ± 21.52	15	10 (66)	7.80 ± 1.55	7.60 ± 1.58	2/10 (20)	2

^#results are expressed as mean ± SD

* P < 0.05, significantly different compared to control value (Fisher´s exact test, two-tail)

 

Table 3: Body and organ weights of females mice exposed to manganese chloride (MnCl₂) for 12 weeks via drinking water^#

Details	Treatments
	Control	1000 mg/l	2000 mg/l	4000 mg/l	8000 mg/l
No. of animals	8	12	12	12	12
Body weight (g)	34.6 ± 6.08	32.84 ± 5.12	33.31 ± 3.40	33.21 ± 3.02	33.33 ± 4.53
Ovarian weights (mg/10g bw)^+	2.12 ± 0.83	2.5 ± 0.74	2.4 ± 0.79	3.50 ± 1.06*	4.70 ± 2.3*
Uterine weights (mg/10g bw)^+	23.60 ± 8.51	31.80 ± 1.28*	34.50 ± 8.12*	35.50 ± 6.9**	33.7 ± 9.7*

^#results are expressed as mean ± SD

⁺Relative weights

* P < 0.05, significantly different compared to control value (Student´s t-test)

** P < 0.005, significantly different compared to control value (Student´s t-test)

Applicant's summary and conclusion

Conclusions:

Although deviations from guideline were made, the study fulfills general scientific requirements, i.e the prolonged exposure duration assures the manifestition of any effects in the P generation despite not dosing during pregnancy. Since the aim of this study was to assess the effects on fertility of the parental animals, the given information are sufficient to assess these effects and are sufficiently documented. Consequently, the results can considered to be reliable and appropriate to assess the effects of manganese acetate on the fertility in mice because MnCl2 can serve as a read-across substance for the former. The NOAEL of manganese chloride was determined to be 8000 mg/l drinking water, which is highest dose tested, and could therefore also be even much higher, for several endpoints attributed to both males and females, e.g. number of implantations. From the effects observed, the NOAELs on the number of pregnant females, number of implantations and number of viable fetuses are considered to be the most relevant ones to assess the effects of Manganese chloride on fertility. The effects on the relative ovarian and uterine weights are mainly attributed to the prolonged exposure to manganese chloride and were shown not to be correlated with the reproductive performance, i.e. fertility, of the animals. Consequently, the NOAELs derived from these effects are considered to be insignificant to assess effects on fertility and can therefore be neglected.
Hence, in order to derive an overall NOAEL of Manganese acetate on fertility from this experimental set-up, the NOAEL can considered to be ≥ 352 mg/kg bw/day (males) and ≥ 358.84 mg/kg bw/day (females), calculated from the average water consumption, whereas the NOAEL of the dams is considered to be the more relevant one for reproductive toxicity. Additionally, the LOAEL can assumed to be 634.92 mg/kg bw/day.
Within this experimental set-up, no relevant adverse effects on reproductive performance at doses ≥ 358.84 mg/kg bw/day Manganese chloride (calculated) could be detected, and the LOAEL of 634.98 mg/kg bw/day also does not refer to relevant endpoints to assess fertility. Additionally, the treatment interval is much longer than the recommended minimum interval, which can also contribute to the severity of the effects. So it can be concluded that Manganese acetate is not or negligibly toxic to reproduction.

Executive summary:

In a screening reproduction study similar to OECD guideline 421, Manganese chloride was administered to 14 male Swiss mice per dose (28 untreated females, Experiment 1) and 15 females (5 untreated males, Experiment 2) in drinking water at dose levels of 0, 1000, 2000, 4000, 8000 mg/l water. At the highest dose level, a significant reduction on the number of pregnant females (treated males) and the number of implantations and viable fetuses (treated females) was observed.

Consequently, the LOAEL is estimated to be 634.92 mg/kg bw/day (calculated from water consumption). The NOAEL was determined to be ≥ 358.84 mg/kg bw/day.

This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.