Registration Dossier
Registration Dossier
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EC number: 211-334-3 | CAS number: 638-38-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Link to relevant study records
- 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.
- 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
- 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) - 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
- Remarks:
- Doses / Concentrations:
1000, 2000, 4000, 8000 mg/l
Basis:
nominal in water - 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 - 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
- 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
- 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: 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
- 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)
- 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)
- 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
- 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
- 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)
- 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
- 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
- Remarks on result:
- other: no information available
- Reproductive effects observed:
- not specified
- 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.
- Endpoint:
- one-generation reproductive toxicity
- 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: see 'Remark'
- Remarks:
- Read-across substance, guideline 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.
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
- Deviations:
- yes
- Remarks:
- Pre-mating dosing time only 28 days
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD) BR
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- 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 10 ppm manganese 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
ENVIRONMENTAL CONDITIONS
- 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 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- 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.
TEST ATMOSPHERE
- 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. - 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 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Daily by optical sensor as described in "Details on exposure"
- 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
- Details on study schedule:
- Litters 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.
- Remarks:
- Doses / Concentrations:
0, 0.05, 0.5, 1 mg/m³ Mn
Basis:
nominal conc.
calculated, referring to manganese - Remarks:
- Doses / Concentrations:
0.001 ± 0.000, 0.157 ± 0.011, 1.50 ± 0.10, 3.03 ± 0.18 mg/m³ MnSO4
Basis:
analytical conc.
based on daily optical sensor data - Remarks:
- Doses / Concentrations:
0, 0.150, 1.53, 3.10 mg/m³ MnSO4
Basis:
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 - Positive control:
- no data
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly
BODY WEIGHT: Yes
- Time schedule for examinations: At least weekly - Oestrous cyclicity (parental animals):
- no data
- Sperm parameters (parental animals):
- no data
- Litter observations:
- STANDARDISATION OF LITTERS
- 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
PARAMETERS EXAMINED
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. - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring (each one mal and female per dam) were sacrificed at 1, 14, 19, 45, 63 days of age.
- These animals were subjected to postmortem examinations as follows:
The following tissues were collected: (n = 1 rat/sex/litter) and analyzed for manganese concentrations: lung, liver, brain, and whole blood. A bone sample was also collected from all pups. Skull cap was collected on PND 1, while femur samples were collected thereafter. In addition, two pups per litter (1 pup/sex/litter) with visible milk bands were selected immediately after the end of the first postnatal inhalation day (PND 1) and a sample of stomach contents containing milk was collected and analyzed for manganese concentration. Brain samples collected from older pups (PND ≥ 19) were dissected into olfactory bulb, striatum, and cerebellum samples. Pancreas samples were also collected from the older pups. All tissue samples were transferred to plastic vials, frozen in liquid nitrogen, and stored at approximately -80°C until manganese analysis was performed.
HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were weighed: Whole body, brain, lung, liver, pancreas - Statistics:
- 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.
- Reproductive indices:
- no data
- Offspring viability indices:
- no data
- Clinical signs:
- no effects observed
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- body weight, see table 1
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- body weight, see table 1
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- not specified
- Other effects:
- not examined
- Description (incidence and severity):
- Test substance intake: not applicable
- Reproductive function: oestrous cycle:
- not specified
- Reproductive function: sperm measures:
- not specified
- Reproductive performance:
- not specified
- Dose descriptor:
- NOAEC
- Remarks:
- highest dose tested
- Effect level:
- 3.1 mg/m³ air (nominal)
- Based on:
- test mat.
- Remarks:
- MnSO4
- Sex:
- female
- Basis for effect level:
- other: clinical signs; mortality; body weight; organ weights;
- Clinical signs:
- no effects observed
- Mortality / viability:
- not specified
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- see table 2
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- see table 2
- Gross pathological findings:
- not specified
- Histopathological findings:
- not specified
- Dose descriptor:
- NOAEC
- Remarks:
- highest dose tested
- Generation:
- F1
- Effect level:
- 3.1 mg/m³ air (nominal)
- Based on:
- test mat.
- Remarks:
- MnSO4
- Sex:
- male/female
- Basis for effect level:
- other: clinical signs; body weight Organ weights: PND 1: Brain, lung, liver PND 14: Lung, liver PND 19: Brain (male), lung, pancreas PND 45 ± 1: Lung, liver, pancreas PND 63 ± 1: Lung, brain, pancreas
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- 1.53 mg/m³ air (nominal)
- Based on:
- test mat.
- Remarks:
- MnSO4
- Sex:
- male/female
- Basis for effect level:
- other: Organ weights: PND 14: Brain PND 19: Brain, liver PND 45 ± 1: Brain
- Reproductive effects observed:
- not specified
- Conclusions:
- Although deviations from guideline were made, the study fulfills general scientific requirements, i.e. dosing of the dams was done constantly in adequate duration prior to mating until lactation, dosing of the pups was done in utero and during lactation, so that all relevant substance-related effects can manifest. Since the aim of this study was to screen the effects on fertility of the parental animals as well as 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 sulphate 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. A NOAEC of 1.53 mg/m³ was only observed in pups on 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. On the other hand, the brain, especially olfactory bulb, was shown to be one of the most affected organs in MnSO4 inhalation studies regarding manganese tissue concentration in adult rats (Dorman et al., NeuroToxicology 26 (2005) 625-632). 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, derived from effects on both dams and pups, is considered to be the relevant one.
Within this experimental set-up, no relevant adverse effects on neither reproductive performance on the P generation nor 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 reproduction. - 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.
There were no relevant compound related toxic effects in the main categories of systemic or reproductive 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 P and F1 generations.
This study is acceptable and satisfies with restrictions the guideline requirement for a one-generation reproductive study (OECD 415) in rats.
Referenceopen allclose all
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
The number of viable fetuses decreased dose-dependently
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)
Inhalation exposure to MnSO4 during gestation and lactation did not affect terminal
maternal (PND 18) body weight (Table 1).
The majority of F0 rats had no observable clinical signs. Clinical signs included alopecia and transient weight loss and were neither clinically relevant nor related to MnSO4 inhalation. Macroscopic lesions (urinary calculi and secondary hydronephrosis or hydroureter) were observed in 16/35 dams. Chemical analysis of several representative calculi revealed that the calculi were composed of magnesium ammonium phosphate (struvite) or calcium oxalate monohydrate. Urinary tract lesions did not demonstrate a dose-response relationship, were seen in control rats, and were deemed to be unrelated to MnSO4 inhalation.
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Only body weight was observed and no MnSO4 related effects were found (see table 1)
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
MnSO4 exposure was kept constant, no relation to body weight was made.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No data
ORGAN WEIGHTS (PARENTAL ANIMALS)
No effects (see table 1)
The majority of F1 rats had no observable clinical signs. Clinical signs included alopecia and transient weight loss and were neither clinically relevant nor related to MnSO4 inhalation. Macroscopic lesions (urinary calculi and secondary hydronephrosis or hydroureter) were observed in 56/70 PND 45 pups, and 48/59 PND 63 pups. Chemical analysis of several representative calculi revealed that the calculi were composed of magnesium ammonium phosphate (struvite) or calcium oxalate monohydrate. Urinary tract lesions did not demonstrate a dose-response relationship, were seen in control rats, and were deemed to be unrelated to MnSO4 inhalation.
BODY WEIGHT (OFFSPRING)
Combined in utero and postnatal inhalation exposure to MnSO4 did not affect neonatal body weight gain between PND 0 and PND 19 in either female ( p = 0.065) or male ( p = 0.374) pups. High-dose MnSO4 exposure (1 mg Mn/m3) was associated with decreased body weights in PND 19 pups (see table 2). Neonatal body weights were also decreased on PND 1 ( p = 0.043) and PND 14 ( p = 0.049); however, post hoc analysis did not reveal a treatment-related effect.
ORGAN WEIGHTS (OFFSPRING)
No effects (see table 2)
Table 1: Maternal (PND 18) organ weights following combined gestational and lactational exposure to air or MnSO4
Organ |
Nominal MnSO4concentration (mg Mn / m³) |
|||
0 |
0.05 |
0.5 |
1 |
|
Brain |
1.97 ± 0.03 |
1.98 ± 0.02 |
1.98 ± 0.04 |
1.90 ± 0.04 |
Lung |
1.47 ± 0.08 |
1.35 ± 0.06 |
1.47 ± 0.08 |
1.36 ± 0.05 |
Liver |
11.86 ± 0.69 |
11.94 ± 0.92 |
11.05 ± 1.14 |
10.22 ± 0.75 |
Pancreas |
0.84 ± 0.08 |
0.79 ± 0.08 |
0.81 ± 0.07 |
0.66 ± 0.05 |
Body weight |
257.3 ± 8.7 |
259.5 ± 11.2 |
257.5 ± 14.9 |
232.4 ± 8.4 |
Group size |
8 |
10 |
9 |
8 |
Note: Data for maternal (PND 18) organ weights (g) are expressed as mean ± SEM
Table 2: Pup organ weights and terminal body weights followingin uteroand postnatal exposure to manganese
PND |
Organ |
Nominal MnSO4concentration (mg Mn / m³) |
|||
0 |
0.05 |
0.5 |
1 |
||
1a |
Body weight |
7.62 ± 0.22 |
7.15 ± 0.16 |
7.64 ± 0.18 |
6.94 ± 0.31 |
Brain |
0.281 ± 0.008c |
0.260 ± 0.011 |
0.299 ± 0.008 |
0.273 ± 0.011 |
|
Lung |
0.124 ± 0.005d |
0.120 ± 0.011 |
0.127 ± 0.005 |
0.120 ± 0.006 |
|
Liver |
0.286 ± 0.010 |
0.275 ± 0.007 |
0.296 ± 0.011 |
0.274 ± 0.013 |
|
Group sizeb |
16 |
20 |
18 |
16 |
|
14a |
Body weight |
28.98 ± 0.84 |
27.90 ± 1.03 |
29.78 ± 1.50 |
24.87 ± 1.35 |
Brain |
1.197 ± 0.015 |
1.192 ± 0.016 |
1.216 ± 0.019 |
1.104 ± 0.036e |
|
Lung |
0.436 ± 0.020 |
0.410 ± 0.011 |
0.437 ± 0.016 |
0.403 ± 0.018 |
|
Liver |
0.830 ± 0.033 |
0.769 ± 0.036 |
0.841 ± 0.056 |
0.689 ± 0.036 |
|
Group sizeb |
16 |
20 |
18 |
16 |
|
19 |
Body weight |
44.96 ± 1.30 |
41.76 ± 1.59 |
44.56 ± 2.12 |
35.99 ± 2.22e |
Brain (male) |
1.49 ± 0.03 |
1.48 ± 0.02 |
1.49 ± 0.03 |
1.40 ± 0.05 |
|
Brain (female) |
1.42 ± 0.02 |
1.43 ± 0.03 |
1.42 ± 0.02 |
1.30 ± 0.05e |
|
Lung |
0.50 ± 0.03 |
0.47 ± 0.02 |
0.53 ± 0.03 |
0.45 ± 0.02 |
|
Liver |
1.85 ± 0.10 |
1.63 ± 0.07 |
1.83 ± 0.13 |
1.33 ± 0.10e |
|
Pancreas |
0.11 ± 0.01 |
0.09 ± 0.01 |
0.11 ± 0.01 |
0.09 ± 0.01 |
|
Group sizeb |
16 |
20 |
18 |
16 |
|
45 ± 1 (female) |
Body weight |
171.2 ± 6.7 |
157.8 ± 6.4 |
162.4 ± 4.5 |
151.4 ± 6.6 |
Brain |
1.85 ± 0.03 |
1.80 ± 0.03 |
1.79 ± 0.03 |
1.73 ± 0.03f |
|
Lung |
1.18 ± 0.04 |
1.09 ± 0.05 |
1.15 ± 0.05 |
1.11 ± 0.06 |
|
Liver |
8.73 ± 0.45 |
8.10 ± 0.51 |
8.26 ± 0.30 |
7.50 ± 0.41 |
|
Pancreas |
0.55 ± 0.06 |
0.58 ± 0.04 |
0.58 ± 0.04 |
0.56 ± 0.05 |
|
Group size |
9 |
10 |
8 |
8 |
|
45 ± 1 (male) |
Body weight |
203.8 ± 15.4 |
198.9 ± 9.1 |
193.5 ± 7.2 |
180.6 ± 12.7 |
Brain |
1.93 ± 0.04 |
1.90 ± 0.03 |
1.90 ± 0.03 |
1.73 ± 0.06e |
|
Lung |
1.28 ± 0.06 |
1.22 ± 0.04 |
1.37 ± 0.09 |
1.23 ± 0.08 |
|
Liver |
10.37 ± 0.71 |
10.04 ± 0.55 |
9.69 ± 0.53 |
9.35 ± 0.72 |
|
Pancreas |
0.60 ± 0.07 |
0.60 ± 0.05 |
0.57 ± 0.03 |
0.57 ± 0.05 |
|
Group size |
7 |
10 |
10 |
8 |
|
Note: Mean (SEM) pup organ weights (g) and terminal body weights (g) followingin uteroand postnatal exposure to manganese.
aNo gender effect noted.
bGroup size reflects one male and one female pup per litter.
cExcludes two outliers
dExcludes one outlier
ep < 0.05
fp = 0.07
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 352 mg/kg bw/day
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Three equivalently reliable Klimisch 2 studies on read-across substances are available, thereunder two performed equivalent to OECD guideline 421, one according OECD 451 which assessed also the effects on reproductive organs and tissues. All studies are sufficiently reliable to cover this endpoint and provide consistent results, i.e. Manganese (II) acetate does not influence to relevant parts fertility and reproductive performance. Consequently, the available data meets fully the tonnage-driven data requirements under REACH, no datagaps were observed.
Since according to REACh Annex IX column 2, "A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data" and the available data allow to draw the conclusion that no effects regarding the toxicity of Manganese (II) acetate to reproduction will be observed in a Pre-natal developmental toxicity study or Two-generation reproductive toxicity study, further studies can be omitted due to animal welfare.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 3.1 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Only one study available on Read-across substance MnSO4, classified as Klimisch 2, which is sufficiently reliable. Furthermore, the study is actually not required since the inhalation route is not a relevant route for humans and the oral route is already covered by three Klimisch 2 studies. Hence, no datagaps are identified and the requirements under REACH are fully met.
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
According to the data requirements as laid down in regulation (EC) 1907/2006, Annex IX, Column 1, Section 8.7.3 a reproductive toxicity study needs to be conducted only if the 28-day or 90-day study indicates adverse effects on reproductive organs or tissues. Concerning repeated dose toxicity, an even more reliable, well documented 2 year chronic / carcinogenicity study according to OECD guideline 451 is available (NTP, 1993). The given data shows no effects of Manganese (II) sulfate monohydrate on reproductive organs in both mice and rats and in both sexes.
In a study similar to OECD 421 on rats (Järvinen, 1795), the NOAEL of manganese was determined to be 1004 mg/kg food, which is highest dose tested, and could therefore also be even much higher. Assuming an average daily food consumption of 60 g/kg bw (Peng 1979), the NOAEL can calculated to be approx. 60.24 mg/kg bw/day manganese, which corresponds to a NOAEL of 189.7 mg/kg bw/day Manganese acetate. Within this experimental set-up, no adverse effects on reproductive performance up to 189.7 mg/kg bw/day Manganese acetate (calculated) could be detected and no LOAEL could be determined, so it can be concluded, based on these results, that Manganese acetate is not toxic to reproduction.
Since it was aimed to determine the NOAEL as precisely as possible, an additional study similar to OECD 421 on mice, was taken into account (Elbetieha, 2001). Although deviations from guideline were made, the study fulfills general scientific requirements, i.e the prolonged exposure duration assures the manifestation 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 be assumed to be 634.92 mg/kg bw/day, based on the pregnancy index (males) and numer of implantations and fetuses’ viability index (females).
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.
Taking into account the inhalative route, another study is available on Manganese sulfate (Dorman, 2005), similar to OECD 415. Although deviations from guideline were made, the study fulfills general scientific requirements, i.e. dosing of the dams was done constantly in adequate duration prior to mating until lactation, dosing of the pups was done in utero and during lactation, so that all relevant substance-related effects can manifest. Since the aim of this study was to screen the effects on fertility of the parental animals as well as 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 sulphate 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. A NOAEC of 1.53 mg/m³ was only observed in pups on 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. On the other hand, the brain, especially olfactory bulb, was shown to be one of the most affected organs in MnSO4 inhalation studies regarding manganese tissue concentration in adult rats (Dorman et al., NeuroToxicology 26 (2005) 625-632). 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/m³is considered as insignificant and can be neglected and a NOAEC of≥3.10 mg/m³, derived from effects on both dams and pups, is considered to be the relevant one.
Within this experimental set-up, no relevant adverse effects on neither reproductive performance on the P generation nor development of the F1 generation could be detected at doses≥3.10 mg/m³MnSO4. So it can be concluded that Manganese acetate is not or negligibly toxic to reproduction.
So in summary, Manganese (II) acetate did not show any relevant effects within the available, reliable studies which would justify a classification as toxic to reproduction.
Since according to REACH Annex IX column 2, "A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data" and the available data allow to draw the conclusion that no effects regarding the toxicity of Manganese (II) acetate to reproduction will be observed in a Pre-natal developmental toxicity study or Two-generation reproductive toxicity study, further studies can be omitted due to animal welfare.
Short description of key information:
Toxicity to reproduction:
- Chronic study oral (feed), Manganese sulfate, rat (F344/N), mice (B6C3F1), m/f (OECD 451): No adverse effects on reproductive organs or tissues significantly over control were detected, NOAEL = 700 mg/kg bw/d (rat, m) / 791 mg/kg bw/d (rat, f) / 1972 mg/kg bw/d (mouse, m) / 2500 mg/kg bw/d (mouse, f) (highest dose tested)
- subchronic study oral (feed), Manganese sulfate, rat (Sprague-Dawley), f (similar to OECD 421): NOAEL = 189.7 (nominal, highest dose tested, element manganese, P, F1)
- subchronic study oral (feed), Manganese chloride, mouse (Swiss), m/f (similar to OECD 421): NOAEL = 352 mg/kg bw/d (males, P, pregnancy index)
- subacute to subchronic study, inhalation (aerosol, whole body), Manganese sulfate, rat (Crl:CD(SD) BR), m/f (similar to OECD 415): NOAEL = 3.1 mg/m³ air (nominal, 6h/d, highest dose tested)
Justification for selection of Effect on fertility via oral route:
Three equivalently reliable Klimisch 2 studies on read-across substances are available, thereunder two performed equivalent to OECD guideline 421. The highest dose tested in the study by Järvinen (1975) was 189.7 mg/kg bw/day manganese acetate (recalculated), where no effects occured. In the study by Elbetieha (2001) higher doses were tested, up to 706.5 mg MnCl2 /kg bw/day, which enable one to draw more concrete conclusions on the NOAEL.
Justification for selection of Effect on fertility via inhalation route:
Only one inhalation study available
Effects on developmental toxicity
Description of key information
Developmental toxicity:
- Chicken embryo test, Single-Comb White Leghorn chicken, single injection of Manganese (II) acetate or MnSO4: NOAEL = 10 mg/egg (teratogenicity, embryotoxicity, Manganese (II) acetate & MnSO4, highest dose tested), which corresponds roughly to ca. 200 mg/kg bw
- subacute to subchronic study, inhalation (aerosol, whole body), Manganese sulfate, rat (Crl:CD(SD) BR), m/f (similar to OECD 415): NOAEL = 3.1 mg/m³ air (nominal, 6h/d, highest dose tested, developmental toxicity)
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- other: experimental result on substance Manganese (II) acetate and analogue
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Testing was performed on non-standard species and no guideline is available for this testing procedure. However, the testing was performed on Manganese (II) acetate itself and additionally on the read-across substance MnSO4, the results and evaluation indicate that the chicken embryo test is capable of demonstrating the teratogenic potential of compounds, as well as the given data lead to the conclusion that the test in general was well-performed. Consequently, the study and obtained results can considered to be reliable.
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- The test substances were applied to the developing chicken embryo (fresh fertile eggs), whereas four test conditions were used: injection via the air cell and via the yolk and at two times, after 0 hr and 96 hr incubation. LD50 values were determined for each test condition. All embryos and hatched chicks were examined grossly for any abnormalities in development, both functional and structural.
- GLP compliance:
- no
- Limit test:
- no
- Species:
- other: chicken
- Strain:
- other: Single-Comb White Leghorn
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: animals were retrieved as fresh fertile eggs from Truslow Farms, Inc., Chestertown, Md.
- Age at study initiation: 0 h and 96 h after fertilization
- Weight at study initiation: no data
- Fasting period before study: not applicable
- Housing: no data
- Diet (e.g. ad libitum): not applicable
- Water (e.g. ad libitum): not applicable
- Acclimation period: not applicable - Route of administration:
- other: injection in either yolk sac or air cell
- Vehicle:
- water
- Details on exposure:
- Test substances up to 10 mg per egg were solved in max. 100 µl water and injected in either yolk sac or air space after 0 h or 96 h of incubation.
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- not applicable
- Duration of treatment / exposure:
- Until death of the embryo or hatching.
- Frequency of treatment:
- once
- Remarks:
- Doses / Concentrations:
up to 10 mg, 5 dose levels
Basis:
other: nominal amount per egg - No. of animals per sex per dose:
- At least 20 embryos for each of the four treatments, resulting in at least 100 embryos for each dose level of each test substance.
- Control animals:
- yes, concurrent no treatment
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Preliminary range-finding study. An attempt was made to use a dose that would be lethal to a significant number of embryos as the highest test level, and a dose that would be a no-effect level with respect to mortality as the lowest test level.
- Maternal examinations:
- not applicable
- Ovaries and uterine content:
- not applicable
- Fetal examinations:
- - External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes
All nonviable embryos and hatched chicks were examined grossly for abnormalities of a structural nature as well as for signs of toxicosis such as edema and hemorrhage. At least five embryos and hatched chicks were randomly selected from the group treated at the highest dose level for examination of the viscera.
Embryos with apparent skeletal defects that could not be readily classified were either X-rayed or cleared and stained with Alizarin Red S to determine the specific anomaly present. All observations were tabulated and analyzed. - Statistics:
- For each test condition, the following analyses were made.
(a) The percentage mortality at each dose level was calculated as the total number of nonviable embryos divided by the total number of treated eggs. These mortality data were examined for a linear relationship between the probit percent mortality versus the logarithm of the dose and, where possible, an LD5O was estimated (Finney, D. J. (1964). Prohit Analysis. 2nd ed.. Apendix I. Cambridge Press, Cambridge, Mass.).
(b) The total number of birds having one or more abnormalities was expressed as a percentage of the total number of eggs treated. Abnormalities included all structural anomalies as well as toxic responses such as edema, hemorrhage, hypopigmentation of the down, significant growth retardation, cachexia, and other nerve disorders.
(c) The total number of birds having a structural abnormality of the head, viscera, limbs, or body skeleton was expressed as a percentage of the total number of eggs treated. Toxic responses and other disorders noted under (b) were excluded. This index was considered the principal measure of the teratogenic potential of the compound.
All of the above indices were analyzed by the Chi2 test (Snedecor, G., Cochran. W. G. (1967). Statistical Methods. 6th ed. Iowa State Univ. Press. Ames.) for significant differences from the solvent background. Additionally, the data on total abnormalities (b) were subjected to regression analysis versus dose as described in (a). - Details on maternal toxic effects:
- Maternal toxic effects:not examined. Remark: not applicable
- Remarks on result:
- other: no information available
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:yes. Remark: Relevance for mammals
Details on embryotoxic / teratogenic effects:
Except for air cell treatment at 96 hr, manganese acetate in water produced no appreciable mortality (up to 10 mg/egg) and no significant terata
were observed under the other test conditions. Air cell treatment at 0 hr resulted in a high incidence of birds with hypopigmentation of the down, an observation considered in these analyses as a toxic response rather than a teratogenic one. However, air cell treatment at 96 hr resulted in a high incidence of birds with severe abnormalities at all three test levels that allowed some to hatch. The defects involved primarily the beak, eyes, and eyelids. - Dose descriptor:
- NOAEL
- Remarks:
- highest level tested
- Effect level:
- 10 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese acetate
- Basis for effect level:
- other: teratogenicity
- Dose descriptor:
- BMD: 25
- Effect level:
- 10 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese acetate
- Basis for effect level:
- other: teratogenicity
- Dose descriptor:
- NOAEL
- Remarks:
- highest level tested
- Effect level:
- 10 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese acetate
- Basis for effect level:
- other: embryotoxicity
- Dose descriptor:
- other: LD50
- Effect level:
- 12.9 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese acetate
- Basis for effect level:
- other: embryotoxicity
- Dose descriptor:
- other: LD50
- Effect level:
- 1.47 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese acetate
- Basis for effect level:
- other: embryotoxicity
- Dose descriptor:
- NOAEL
- Remarks:
- highest level tested
- Effect level:
- 10 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese sulfate
- Basis for effect level:
- other: teratogenicity
- Dose descriptor:
- NOAEL
- Remarks:
- highest level tested
- Effect level:
- 10 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese sulfate
- Basis for effect level:
- other: embryotoxicity
- Dose descriptor:
- other: LC50
- Effect level:
- 8.1 other: mg / egg
- Based on:
- test mat.
- Remarks:
- Manganese sulfate
- Basis for effect level:
- other: embryotoxicity
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- Although the testing was performed on non-standard species and no guideline is available for this testing procedure, the results and evaluation indicate that the chicken embryo test is capable of demonstrating the teratogenic potential of compounds, as well as the given data lead to the conclusion that the test in general was well-performed. Additionally, the testing was performed on Manganese (II) acetate itself and furthermore on the read-across substance MnSO4, which broadens the spectrum of the insight in manganese toxicity / teratogenicity. So the study was classified as reliable with restrictions and consequently the results can considered to be reliable.
The given effect levels were not expressed in standard terms such as mg/kg bw but in mg / egg, so that the comparison to other developmental studies is hindered. Unfortunately, the given levels cannot be easily converted in standard levels, because the average weight of an egg can only be estimated to be between 40 – 70 g and additionally the relative weight and distribution of the embryo, which varies over the time, must be taken also into account. Assuming uniform distribution, as it is most likely, the NOAEL in mg/kg bw can roughly estimated to be 20 times higher, as also no resorption issues have to be considered.
The read-across substance MnSO4 showed no teratogenic effects at all four modes of applications and also only minor embryotoxic effects at only one mode of application, leading to the conclusion that manganese itself has no teratogenic effects at all. Also, manganese (II) acetate did not show teratogenic effects in three out of four modes of application, i.e. when injected into the yolk sac and injected in the air cell after 0 h of incubation. The most likely reason for these effects can assumed to be modification in the eggs membrane due to the acidic properties of acetate anion, which leads to a hindered oxygen uptake. As the embryos oxygen demand increases during development, these effects are predominant in the later (96 h) development stages. So, the observed effects are most likely not to be due to the intrinsic possible hazards of manganese (II) acetate but to its acidic properties in combination with the mode of application, which is not relevant for humans or mammals in general.
Consequently, manganese (II) acetate does not need to be considered as developmentally toxic. - Executive summary:
In a developmental toxicity study on chicken embryos, Manganese (II) acetate and Manganese sulfate (aqueous solution) was injected in either the yolk sac or air cell after 0 h or 96 h of incubation of at least 20 fresh fertile eggs from Single-Comb White Leghorn chickens per mode of incubation and dose level at at least five different dose levels up to 10 mg / egg. There were only minor embryotoxic (lethal) effect observed with both substances. MnSO4 did not exhibit any teratogenic effects, and Manganese (II) acetate did also not show teratogenic effects except for the injection into the air cell after 96 h, were abnormalities were mainly observed at the beak, eyes and eyelids. However, these effects are not considered to be relevant for mammals, and therefore the NOAEL was determined to be 10 mg / egg for both Manganese (II) acetate and MnSO4, which was the highest level tested, and therefore no LOAEL could be established.
The developmental toxicity study on chicken embryos is classified as acceptable, suitable for the assessment of teratogenic effects and reliable with restrictions and satisfies general scientific requirements.
- Endpoint:
- developmental toxicity
- 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: see 'Remark'
- Remarks:
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: OECD Guideline 415 (One-Generation Reproduction Toxicity Study)
- Deviations:
- yes
- Remarks:
- Pre-mating dosage time only 28 days
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- other: Crl:CD(SD) BR
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- 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
ENVIRONMENTAL CONDITIONS
- 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 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- 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.
TEST ATMOSPHERE
- 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:
- yes
- 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
- Remarks:
- Doses / Concentrations:
0, 0.05, 0.5, 1 mg/m³ Mn
Basis:
nominal conc.
calculated, referring to manganese - Remarks:
- Doses / Concentrations:
0.001 ± 0.000, 0.157 ± 0.011, 1.50 ± 0.10, 3.03 ± 0.18 mg/m³ MnSO4
Basis:
analytical conc.
based on daily optical sensor data - Remarks:
- Doses / Concentrations:
0, 0.150, 1.53, 3.10 mg/m³ MnSO4
Basis:
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:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least weekly
BODY WEIGHT: Yes
- 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.
STANDARDISATION OF LITTERS
- 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
PARAMETERS EXAMINED
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. - Statistics:
- 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.
- Details on maternal toxic effects:
- Maternal toxic effects:no effects
- Dose descriptor:
- NOAEC
- Remarks:
- highest dose tested
- Effect level:
- 3.1 mg/m³ air
- Based on:
- test mat.
- Remarks:
- MnSO4
- Basis for effect level:
- other: maternal toxicity
- Dose descriptor:
- NOAEC
- Remarks:
- highest dose tested
- Effect level:
- 3.1 mg/m³ air
- Based on:
- test mat.
- Remarks:
- MnSO4
- Basis for effect level:
- other: developmental toxicity
- Dose descriptor:
- NOAEC
- Effect level:
- 1.53 mg/m³ air
- Based on:
- test mat.
- Remarks:
- MnSO4
- Basis for effect level:
- other: other:
- 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. - Remarks on result:
- other: no information available
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- 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.
Referenceopen allclose all
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 200 mg/kg bw/day
- Species:
- other: Chicken embryo
- Quality of whole database:
- No study on Developmental toxicity via the oral route of Manganese (II) acetate is available. However, the chicken embryo test is a suitable alternative in general to demonstrate the teratogenic potential of compounds, and was assessed with Klimisch 2. This classification meets the quality requirements under REACH and is hence sufficient to cover this endpoint. Additionally, the classification of Manganese (II) acetate as not developmentally toxic is in congruence with the results of the testing via inhalation route and other observations made in studies examining toxicity to reproduction, such as the study by Järvinen (1975), which also showed no indication of developmental toxicity. Consequently, the available data meets fully the tonnage-driven data requirements under REACH, no datagaps were observed.
Since according to REACh Annex IX column 2, "A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data" and the available data allow to draw the conclusion that no effects regarding the developmental toxicity of Manganese (II) acetate will be observed in a Pre-natal developmental toxicity study or Two-generation reproductive toxicity study, further studies can be omitted due to animal welfare.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEC
- 3.1 mg/m³
- Study duration:
- subchronic
- Species:
- rat
- Quality of whole database:
- Only one study available on Read-across substance MnSO4, classified as Klimisch 2, which is sufficiently reliable. Furthermore, the study is actually not required since the inhalation route is not a relevant route for humans, but is considered as very useful to confirm the conclusion not to classify Manganese (II) acetate as developmentally toxic by a similar outcome. Hence, no datagaps are identified and the requirements under REACH are fully met.
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Additional information
No study on developmental toxicity via oral route is available. However, a chicken embryo test is available (Verrett, 1980), and although the testing was performed on non-standard species and no guideline is available for this testing procedure, the results and evaluation indicate that the chicken embryo test is capable of demonstrating the teratogenic potential of compounds, as well as the given data lead to the conclusion that the test in general was well-performed. Additionally, the testing was performed on Manganese (II) acetate itself and furthermore on the read-across substance MnSO4, which broadens the spectrum of the insight in manganese toxicity / teratogenicity. So the study was classified as reliable with restrictions and consequently the results can be considered to be reliable.
The given effect levels were not expressed in standard terms such as mg/kg bw but in mg / egg, so that the comparison to other developmental studies is hindered. Unfortunately, the given levels cannot be easily converted in standard levels, because the average weight of an egg can only be estimated to be between 40 – 70 g and additionally the relative weight and distribution of the embryo, which varies over the time, must be taken also into account. Assuming uniform distribution, as it is most likely, the NOAEL in mg/kg bw can roughly estimated to be 20 times higher, as also no resorption issues have to be considered. So the NOAEL was set as 200 mg/kg in order to meet the IUCLID 5 data format requirement.
The read-across substance MnSO4 showed no teratogenic effects at all four modes of applications and also only minor embryotoxic effects at only one mode of application, leading to the conclusion that manganese itself has no teratogenic effects at all. Also, manganese (II) acetate did not show teratogenic effects in three out of four modes of application, i.e. when injected into the yolk sac and injected in the air cell after 0 h of incubation. The most likely reason for these effects can assumed to be modification in the eggs membrane due to the acidic properties of acetate anion, which leads to a hindered oxygen uptake. As the embryos oxygen demand increases during development, these effects are predominant in the later (96 h) development stages. So, the observed effects are most likely not to be due to the intrinsic possible hazards of manganese (II) acetate but to its acidic properties in combination with the mode of application, which is not relevant for humans or mammals in general. Consequently, based on the results of this study, manganese (II) acetate does not need to be considered as developmentally toxic.
The study for developmental toxicity via the inhalation route (Dorman, 2005) 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/m³is considered as insignificant and can be neglected and a NOAEC of≥3.10 mg/m³is 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/m³MnSO4. So it can be concluded that Manganese acetate is not or negligibly toxic to development.
The conclusion derived from these two studies, that a classification of Manganese (II) acetate as non-developmentally toxic is justified, is additionally confirmed by the study by Järvinen (1975), which also showed no indication of developmental toxicity in the F1 generation when assessing the toxicity to reproduction.
So in summary, Manganese (II) acetate does not need to be classified as developmentally toxic, which was confirmed by three Klimisch 2 studies and so the tonnage-driven data requirements under REACH are fully met, no datagaps were observed.
Since according to REACH Annex IX column 2, "A decision on the need to perform a study at this tonnage level or the next on a second species should be based on the outcome of the first test and all other relevant available data" and the available data allow to draw the conclusion that no effects regarding the developmental toxicity of Manganese (II) acetate will be observed in a Pre-natal developmental toxicity study or Two-generation reproductive toxicity study, further studies can be omitted due to animal welfare.
Justification for selection of Effect on developmental toxicity: via oral route:
No study on Developmental toxicity via the oral route of Manganese (II) acetate is available. However, the chicken embryo test is a suitable alternative in general to demonstrate the teratogenic potential of compounds, and was assessed with Klimisch 2.
Additionally, the study by Järvinen (1975), which could not be selceted here due to limitations of the IUCLID 5 program, did not detect any relevant adverse effects in the F1 generation.
Justification for selection of Effect on developmental toxicity: via inhalation route:
Only study available on developmental toxicity via inhalation route.
Justification for classification or non-classification
No substance-related effects regarding toxicity to reproduction or developmetal toxicity were observed, and so Manganese (II) acetate does not need to be classified as toxic to reproduction, neither according to Regulation 1272/2008/EC nor Directive 67/548/EEC.
Additional information
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