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Developmental toxicity / teratogenicity

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developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Read-across substance, OECD guideline 415 followed with deviations, not all stipulated examinations were performed, no raw data provided; however, the given data indicate that the study was well-performed and sufficient to fulfill the requirements of this endpoint, additionally because the deviations from OECD guideline 415 can be assumed to have minor or no impact on th development of the fetuses.
Reason / purpose for cross-reference:
reference to same study

Data source

Reference Type:
Tissue Manganese Concentrations in Lactating Rats and Their Offspring Following Combined in Utero and Lactation Exposure to Inhaled Manganese Sulfate
Dorman DC, McElveen, AM, Marshall MW, Rarkinson CU, James RA, Struve MF, Wong BA
Bibliographic source:
TOXICOLOGICAL SCIENCES 84, 12–21 (2005); doi:10.1093/toxsci/kfi060

Materials and methods

Test guideline
equivalent or similar to guideline
other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
Pre-mating dosage time only 28 days
GLP compliance:
not specified
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
Manganese sulphate
EC Number:
EC Name:
Manganese sulphate
Cas Number:
Molecular formula:
manganese (2+) sulfate
Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Manganese sulfate; Manganese (II) sulfate monohydrate
- Molecular formula (if other than submission substance): MnSO4*H2O
- Physical state: solid, white to pale pink crystalline powder
- Analytical purity: <98%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components: contains 32% Mn by weight
Supplier: Aldrich Chemical Company, Inc. (Milwaukee, WI):

Test animals

other: Crl:CD(SD) BR
Details on test animals or test system and environmental conditions:
- Source: Crl:CD1(SD) BR rats were purchased from Charles River Laboratories, Inc (Raleigh, NC).
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) determined, but not given
- Fasting period before study: no data
- Housing: housed in CIIT’s animal facility, which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Except during inhalation exposure and breeding periods, male and female F0 rats were individually housed in polycarbonate cages containing cellulose fiber chip bedding (ALPHA-driTM; Shepherd Specialty Papers, Kalamazoo, MI). Animal breedings (typically 1 male: 1 female) were conducted overnight in polycarbonate cages containing cellulose fiber chip bedding. All weaned pups were housed with littermates of the same gender in polycarbonate cages (<3 rats/cage) containing cellulose fiber chip bedding.
- Diet (e.g. ad libitum): A pelleted, semipurified AIN-93G certified diet from Bio-Serv (Frenchtown, NJ) formulated to contain approximately 10ppmmanganese and 35 ppm iron was given throughout the study. Food was available to all animals ad libitum except during inhalation exposures.
- Water (e.g. ad libitum): Reverse osmosis purified water containing 50.222–0.546 mg Mn/l was available ad libitum
- Acclimation period: Approx. 2 weeks

- Temperature (°C): 17–23°C
- Humidity (%): 30–70%
- Air changes (per hr): air flow rate sufficient to provide 10–15 air changes per h
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled by automatic controls (lights on approximately 07:00–19:00), i.e. 12h dark : 12h light

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
unchanged (no vehicle)
Details on exposure:
- Exposure apparatus: 8-m3, stainless steel and glass inhalation exposure chambers (Lab Products, Maywood, NJ)
- Method of holding animals in test chamber: Rats were exposed in stainless steel wire cage units; the exposure chamber used to simultaneously expose an individual dam and its litter is described in Vitarella, D.,Wong,B. A., James, R. A., Miller, K. L., Struve,M.F., and Dorman, D. C. (1998). Development of an inhalation system for the conduct of developmental neurotoxicity studies. Inhal. Toxicol. 10, 1095–1117.
- Source and rate of air: MnSO4 atmospheres were generated and characterized using methods described by Dorman, D. C., Struve, M. F., James, R. A., Marshall, M. W., Parkinson, C. U., and Wong, B. A. (2001). Influence of particle solubility on the delivery of inhaled manganese to the rat brain: Manganese sulfate and manganese tetroxide pharmacokinetics following repeated (14-day) exposure. Toxicol. Appl. Pharmacol. 170, 79–87. Airflow through each dam/pup exposure cylinder was controlled by an adjustable metering valve and was maintained at approximately 2.5–3.5 l/min
- Method of conditioning air:
- System of generating particulates/aerosols:
- Temperature, humidity, pressure in air chamber: Chamber temperatures were maintained at 22 ± 4 °C while the relative humidity was maintained at 40 to 60 %.
- Air flow rate: approximately 2.5–3.5 l/min
- Air change rate: approximately 35 to 50 air changes per h
- Method of particle size determination: optical particle sensor
- Treatment of exhaust air: no data
- Other: Animal positions within the exposure chambers were rotated during the experiment to minimize experimental error due to any undetected differences in the environment or the manganese aerosol concentration.

- Brief description of analytical method used: The overall means (±SD) for the chamber concentrations based on daily optical particle sensor data were 0.001 ± 0.000, 0.157 ± 0.011, 1.50 ± 0.10, and 3.03 ± 0.18 mg/m3 for the target exposure concentrations of 0, 0.15, 1.53, and 3.10 mg MnSO4/m3, corresponding to 0, 0.05, 0.5, and 1 mg Mn/m3, respectively. The particle size distribution was 1.03 mm geometric mean diameter (GMD) and 1.52 geometric standard deviation (GSD), 1.05 mm GMD (GSD = 1.53), and 1.07 mm GMD(GSD = 1.55) for the target concentrations of 0.150, 1.53, and 3.10 mg MnSO4/m3, respectively. Control groups were exposed to HEPA-filtered air only. The particle size distribution for the control chamber was 0.79 mm GMD (GSD = 1.52), and particles in the control chamber likely represent dander, feed, and other particulate sources.
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Daily by optical sensor as described in "Details on exposure"
Details on mating procedure:
- M/F ratio per cage: 1/1, no change in mating partners
- Length of cohabitation: up to 14 days until pregnancy
- Proof of pregnancy: vaginal plug and sperm in vaginal smear referred to as day 0 of pregnancy
- No second mating attempt
- After successful mating each pregnant female was caged: individually
Duration of treatment / exposure:
Male F0 rats: 28 days (pre-breeding period) + 14 days mating period (42 days)
Female F0 rats: 28 days (pre-breeding period) + up to 14 days mating period + 19 days pregnancy + 18 days lactation period (66-79 days)
Litters: 19 days
Frequency of treatment:
6 h/day, 7 days/week
Duration of test:
up to 124 days
Doses / concentrationsopen allclose all
Doses / Concentrations:
0, 0.05, 0.5, 1 mg/m³ Mn
nominal conc.
calculated, referring to manganese
Doses / Concentrations:
0.001 ± 0.000, 0.157 ± 0.011, 1.50 ± 0.10, 3.03 ± 0.18 mg/m³ MnSO4
analytical conc.
based on daily optical sensor data
Doses / Concentrations:
0, 0.150, 1.53, 3.10 mg/m³ MnSO4
nominal conc.
No. of animals per sex per dose:
10 males, 10 females of F0 generation
5 male, 5 female litters of each F0 female
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: no data
- Rationale for animal assignment (if not random): Randomization of animals to treatment groups occurred prior to the start of the inhalation exposure and was based upon a weight randomization procedure

Litters for postnatal observations were selected so that they contained at least 5 male and 5 female rats, and the litters were randomly reduced to four animals per sex on PND 4 (postnatal day). Pups were euthanized on PND 1, 14, 19, 45 ± 1, and 63 ± 1, and tissue manganese concentrations determined at these time points. Necropsies on PND 1, 14, and 19 were performed immediately after the end of the 6-h inhalation exposure. Dams were killed on PND 18, and tissue manganese concentrations were subsequently evaluated. Male F0 rats were killed by CO2 after the 2-week breeding period, and tissues were not collected from these animals.


Maternal examinations:

- Time schedule: At least weekly

- Time schedule for examinations: At least weekly
Ovaries and uterine content:
Not examined in general.
Litters were given birth and fed via lactation.
Uteri from nonpregnant rats were collected and stained with potassium ferricyanide to confirm that implantation had not occured.
Fetal examinations:
Not fetuses but born pups were examined.

- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter); each 1 male and female pup was necropsied on PND 1

The following parameters were examined in F1 offspring: Average male and female pup body weights were determined for each litter on PND 0, 7, and 14. Individual male and female pup body weights were measured and recorded at least weekly thereafter. All animals had individual body weights determined at necropsy.
The data for quantitative, continuous variables were compared for the exposure and control groups by tests for homogeneity of variance (Levene’s test), analysis of variance (ANOVA), and Dunnett’s multiple comparison procedure for significant ANOVA. In the event the Levene’s test was significant, the data were transformed using a natural log (ln) transformation. If the Levene’s test was significant following transformation, then the original data were analyzed using nonparametric statistics (Wilcoxon or Kruskal-Wallis). Individual data that appeared to be outliers were critically evaluated using a Dixon-type test for discordancy for an upper outlier. Data collected from neonatal rats were analyzed using an analysis of covariance (using a standard least squares model) to adjust for possible effects associated with the gender of the animal. The data for male and female rats were subsequently pooled when gender effects were not observed. Statistical analyses were performed using JMP software from SAS Institute Inc. (Cary, NC). A probability value of 0.01 was used for Levene’s test, while p<0.05 was used as the critical level of significance for all other statistical tests. Unless otherwise noted, data presented are mean values 6 standard error of the mean (SEM) and reflect comparisons with age-matched air-exposed controls.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Effect levels (maternal animals)

open allclose all
Dose descriptor:
highest dose tested
Effect level:
3.1 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
highest dose tested
Effect level:
3.1 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Dose descriptor:
Effect level:
1.53 mg/m³ air
Based on:
test mat.
Basis for effect level:
other: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: Postnatal examination

Details on embryotoxic / teratogenic effects:
Litters were not observed as fetuses ex utero but postpartum.

Effect levels (fetuses)

Remarks on result:
other: no information available

Fetal abnormalities

not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Although deviations from guideline 415 were made, the study fulfills general scientific requirements to assess the possible teratogenic effects because the deviation from guideline 415 can be assumed to have minor or no impact on the outcome of the test because the dams were constantly dosed during pregnancy and also sufficiently before.
Since the aim of this study here was to screen the effects on the development of the pups, 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 rats because MnSO4 can serve as a read-across substance for the former.
The NOAEC of manganese sulfate was determined to be 3.1 mg/m³, which is highest dose level tested, and could therefore also be even much higher, for most endpoints attributed to both dams and pups including all endpoint which are used to assess developmental toxicity, i.e. all effects observed on the newborn pups which were only dosed via the dams.
A NOAEC of 1.53 mg/m³ was only observed in pups on post natal day (PND) 14, 19 and 45 and its basis is a decreased brain weight. This effect can considered to be of minor importance for the assessment of the developmental toxicity, because on the one hand this effect is only transient and not permanent as PND 63 pups show normal brain weights compared to control. Additionally, PND 1 pups did not show a significantly decreased brain weight. So it can be concluded that the decreased brain weight cannot be attributed to manganese uptake in utero but to the intake via inhalation. As a consequence, the NOAEC of 1.53 mg/m³ is considered as insignificant and can be neglected and a NOAEC of ≥ 3.10 mg/m³ is considered to be the relevant one.
Within this experimental set-up, no relevant adverse effects on the development of the F1 generation could be detected at doses ≥ 3.10 mg/m³ MnSO4. So it can be concluded that Manganese acetate is not or negligibly toxic to development.
Executive summary:

In an one-generation reproduction study (equivalent to OECD 415 with deviations), Manganese sulphate was administered to 10 Crl:CD(SD)BR rats per sex and dose by inhalation of 0, 0.15, 1.53 and 3.1 mg/m³ over 6h/day, 7days/week. Additionally, each 5 males and females per litter were exposed with the dam. The effects of MnSO4 on the newborn pups are considered to be sufficient to assess its teratogenic effects.

There were no relevant compound related toxic effects in the main categories of systemic, reproductive or developmental toxicity evaluated. No relevant LOAEC could be observed. The NOAEC was determined from the highest dose level tested as 3.1 mg/m³ for both dams or offspring.

This study is acceptable and satisfies with restrictions the guideline requirement for a one-generation reproductive study (OECD 415) in rats which also allows the assessment of the teratogenic effects of Manganese (II) acetate.