OligoActiv EDDHA-Mn

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

There is no data available for the registered substance on reproductive toxicity. However, there is data available for the source substances FeNaEDDHMA and manganese salts. The values for the manganese salts were converted to the registered substance under consideration of the maximum percentage of manganese included in the substance. This data is used within a frame of a weight-of-evidence approach to assess the toxicity of the target substance. The whole dataset included in the weight-of evidence approach is shown under 'Additional information'. Based on this the key value for manganese was chosen from a reproduction screening study (Järvinen et al. 1975). The NOAEL was determined to be 803.2 mg/kg bw/day for the manganese moiety of the target substance based on the absence of effects in the highest test dose.

In the one-generation reproduction toxicity study performed with the test item Fe(Na)EDDHMA in rats (NOTOX B.V., 1997) the NOAEL for reproductive performance/fertility was established at 200 mg/kg bw/day based on a slight decrease in the conception indices and a minimal delay in precoital time noted at the high dose level of 750 mg/kg bw/day. Further information can be found under 'Additional information'.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 750 mg/kg bw/day group hunched posture, piloerection and emaciated and/or pale appearance were noted. A few males of the 200 mg/kg bw/day group showed hunched posture.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the 750 mg/kg bw/day group, 1/28 males and 4/28 females died during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain was decreased in animals of both sexes at 750 mg/kg bw/day and in males at 200 mg/kg bw/day. This decrease was dose-related. During the lactation period, body weight gain of females at 750 mg/kg bw/day showed a marked increase. Body weight ratios were decreased during the first 5 weeks of treatment for males and during the first 3 weeks for females at 750 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was decreased in animals of both sexes at 750 mg/kg bw/day and in males at 200 mg/kg bw/day. Food consumption of males receiving 50 mg/kg/day and of females receiving 50 or 200 mg/kg/day remained in the same range as controls, before and after correction for the body weight.
Food consumption was decreased with statistical significance compared to controls, in males receiving 200 mg/kg/day from day 57 of treatment until termination and in males receiving 750 mg/kg/day from the beginning until the end of treatment. Following correction for the body weight, relative food consumption of males receiving 750 mg/kg/day showed a statistically significant decrease in comparison with control males between days 1 to 36 of treatment.
The food consumption of females treated at 750 mg/kg/day was noted as decreased during the premating period and until the end of the gestation period. During the period of lactation the food consumtion of females treated at 750 mg/kg/day was similar as control females. Relative food consumption was decreased during the premating period and during the first week of the gestation period only. In all cases the difference with control females was statistically significant.

Organ weight findings including organ / body weight ratios:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

There was no histopathological evidence of toxicity or infertility.

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

A slight delay of the precoital time in the 750 and 200 mg/kg dose groups was observed.
For all males and females that were paired, mating could be confirmed, resulting in 100 percent mating for each dose group. The fertility and conception indices of the 200 and 50 mg/kg dose groups were comparable with the control group. In the 750 mg/kg dose group these indices were considered to be slightly low, although the difference with controls did not achieve a level of statistical significance.
The gestation index was 100% in all treatment groups and were not affected by treatment with FeEDDHMANa.
In the absence of concomitant histopathological findings, a slight decrease of the fertility and conception indices was noted at 750 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity: based on clinical signs, incidences of mortality, and changes in body weight and food consumption at higher dose levels

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: reproductive toxicity, fertility: based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day.

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no unexpected clinical signs seen among pups of any dose group. Signs that were more frequently observed among the pups of all treatment groups during the first litter check (FLC), post partum phase and last litter check (LLC), consisted of hypothermia, no milk in the stomach and small appearance. These signs normally precede the death of non-viable pups and were considered not to represent a distinct toxic effect caused by the test substance.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Adverse effects on the F1-offspring consisted of an increased mortality rate during postnatal days 0-4 seen across the treatment groups (please refer to the table 1 in the section "Any other information on results incl.tables". The majority of the post natal loss was attributable to a few litters of each group, including 1, 3 and 3 litters in the low, mid and high dose groups, respectively. For further details, please refer to the table 2 in the section "Any other information on results incl. tables".

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reduced body weights were noted during days 4 to 21 of lactation in pups of the high dose group (750 mg/kg bw/day).

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test item related macroscopic findings.

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

no

Reproductive effects observed:

not specified

Comparison of the relative number of live and dead pups during the various phases of lactation (i.e. first litter check, post partum days 0-4 and post partum days 5-21) revealed a statistically significant increase in post natal loss during days 0-4 in all treatment groups. The Viability Indices of these groups were correspondingly low. However, it was noted that the number of pups found dead at the first litter check (FLC) of control dams, were relatively high in comparison with the treatment groups. In accordance with these findings a relatively low Live Birth Index was noted in control litters. The following number of decedents among the F1-offspring was noted when calculating the total number of deaths during days 0-4 Post partum including the FLC:

Table 1: TOTAL DEAD PUPS DURING FLC & DAYS 0-4 P.P.

	GROUP 1 0 MG/KG	GROUP 2 50 MG/KG	GROUP 3 200 MG/KG	GROUP 4 750 MG/KG
Litters affected	5	8	9	6
Total dead pups	21	24	44	42
Mean	0.9	0.9	1.7	2.2
N (litters)	24	27	26	19

When comparing the total number of dead pups over the first 4 days of lactation, including the FLC, the mean number of dead pups per litter in the 50 mg/kg dose group equals the number in the control group. At the 200 and 750 mg/kg dose level, the mean number of deaths per litter revealed a dose-related increase when compared to the control group. A similar pattern was seen in the Overall Surviving Indices of the treatment and control groups. However, it must be considered that 2 females of the 750 mg/kg dose group died on the first day of lactation and that therefore 26 of 29 of their pups were killed in extremis.

After culling of all litters to 4 male and 4 female pups (if practically possible), the low mortality rate seen among pups of the treatment groups was comparable to that of the control group. This was also reflected by the comparable Weaning indices in control and treated groups.

Table 2: POSTNATAL LOSS AND VIABILITY
Endpoint	Dose Group [mg/kg bw/day]
	0	50	200	750
Dams with total litter loss at FLC [n]	1	0	0	0
(Pups lost [n])	(13)
Dead pups at FLC [n]	19	3	5	3
(Litters [n])	(5)	(3)	(3)	(1)
Postnatal loss PND 0-4 [n]	2	21	39	39
(Litters [n])	(1)	(7)*	(7)	(6)
Viability index (%)	99.4	94.9	90.4	85.4
FLC: first litter check
*: including 1 female with total pup loss (15/15)

CHEMICAL ANALYSIS

For the nominal concentrations of 0, 10, 40 and 150 mg/g, the concentrations analysed were in agreement with the concentrations prepared in this study. The test item was found to be mixed homogeneously through the vehicle and to be stable for 4 hours at ambient temperature.

Conclusions:

Based on the mortality observed in animals of the high dose group, and the reduced body weight gain and food consumption seen in animals of the mid- and high-dose groups, a parental No Observed Adverse Effect Level (NOAEL) of 50 mg/kg was established. Due to the increased post natal loss and reduced viability index in the treatment groups observed on post natal Days 0-4, the developmental NOAEL could, in fact, not be established. However, based on the results of the 28-day and 90-day study with FeEDDHMANa, it cannot be excluded that anaemia and/or impaired renal function may have been present that finally caused the observed litter losses. On the other hand, the finding in the low dose group consisted of only one female with total litter loss (15 pups) and 6 other females with 1 dead pup/litter which was within normal limits and might have occurred by chance. Therefore, a developmental NOAEL of 50 mg/kg might be considered; effects at higher levels were considered to be closely related to (subclinical) maternal toxicity. The same result is expected for the target substance since the nature of the constituents of the source substance is the same and they act via the same mechanism. No differences in the magnitude of the effects are expected. Therefore the same NOAEL is also applicable for the target substance.

Executive summary:

In an one-generation reproduction toxicity study FeEDDHMANa in distilled water was administered to 28 Wistar rats/sex/dose level by single oral gavage (5 mL/kg bw) at dose levels of 50, 200 or 750 mg/kg bw/day. A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanised as soon as possible after weaning.

The primary effect of treatment with the test item on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxcity in parental animals was 50 mg/kg bw/day under the conditions of this study.

In the offspring, increased post natal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. With special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was 50 mg/kg bw/day under the conditions of this study.

Based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day, the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day.

This study is acceptable and satisfies the guideline requirement for a one-generation reproduction toxicity study (OECD 415).

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Description (incidence and severity):

The majority of the maternal rats had no observable clinical signs. Clinical signs included alopecia and transient weight loss and were neither clinically relevant nor related to MnSO4 inhalation.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Inhalation exposure to MnSO4 during gestation and lactation did not affect terminal maternal (PND 18) body weight (Table 1).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MnSO4 exposure was kept constant, no relation to body weight was made.

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Dose descriptor:

NOAEC

Effect level:

15.03 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: highest dose tested

Critical effects observed:

no

Clinical signs:

no effects observed

Description (incidence and severity):

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.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not specified

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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 (see table 2)

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

High-dose manganese exposure (1 mg Mn/m3) was associated with decreased brain weights in PND 14 pups, PND 19 female pups, and PND 45 +- 1 male pups. Analysis of absolute brain weight (relative to body weight) revealed a slight increase in relative brain weights in PND 19 pups occurred. Mean relative brain weights in air-exposed and high-dose MnSO4-exposed PND 19 pups were 3.3 +- 0.09 and 3.9 +- 0.17%, respectively. No gender effect on relative brain weight was observed in the PND 19 pups. Relative brain weights in all other treatment groups were unaffected by MnSO4 exposure. High-dose manganese exposure (1 mg Mn/m3) was also associated with decreased liver weight in PND 19 pups. Male PND 63 pups exposed to MnSO4 at 0.5 mg Mn/m3 had decreased liver weights (13.72 +- 1.07 g) when compared with age-matched air-exposed controls (17.10 +- 0.58 g). All other organ weights measured on PND 63 +-1 were unaffected by MnSO4 exposure (table 2).

Gross pathological findings:

not specified

Histopathological findings:

not specified

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

15.03 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

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

Remarks on result:

other: highest dose tested

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

7.42 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

Sex:

male/female

Basis for effect level:

other: Organ weights: PND 14: Brain PND 19: Brain, liver PND 45 ± 1: Brain

Critical effects observed:

no

Reproductive effects observed:

no

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
1^a	Body weight	7.62 ± 0.22	7.15 ± 0.16	7.64 ± 0.18	6.94 ± 0.31
	Brain	0.281 ± 0.008^c	0.260 ± 0.011	0.299 ± 0.008	0.273 ± 0.011
	Lung	0.124 ± 0.005^d	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 size^b	16	20	18	16
14^a	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 ± 0.036^e
	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 size^b	16	20	18	16
19	Body weight	44.96 ± 1.30	41.76 ± 1.59	44.56 ± 2.12	35.99 ± 2.22^e
	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 ± 0.05^e
	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 ± 0.10^e
	Pancreas	0.11 ± 0.01	0.09 ± 0.01	0.11 ± 0.01	0.09 ± 0.01
	Group size^b	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 ± 0.03^f
	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 ± 0.06^e
	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

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.
The NOAEC of manganese sulphate was determined to be 3.1 mg/m³, which is highest dose level tested and corresponds to 15.03 mg/m³ for manganese moiety in the target substance and could therefore also be even much higher, for most endpoints attributed to both dams and pups. A NOAEC of 1.53 mg/m³, which corresponds to 7.42 mg/m³ for manganese moiety in the target substance, 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 1.52 mg/m³ (MnSo4) is considered as insignificant and can be neglected and a NOAEC of ≥ 3.10 mg/m³ (MnSO4), derived from effects on both dams and pups, is considered to be the relevant one. This corresponds to 15.03 mg/m³ for the manganese moiety of the target substance.
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 ≥ 15.03 mg/m³ for manganese moiety in the target substance. So it can be concluded that Manganese 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 (MnSO4) for both P and F1 generations. This corresponds to 15.03 mg/m³ for the manganese moiety of the target substance.

This study is acceptable and satisfies with restrictions the guideline requirement for a one-generation reproductive study (OECD 415) in rats.

Reference 3

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Only females were exermined and no substance-related or any other effects on body weight were observed

Food consumption and compound intake (if feeding study):

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

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.

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
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.

Dose descriptor:

NOAEL

Effect level:

ca. 2 088 mg/kg bw/day

Based on:

test mat.

Remarks:

converted to target substance

Sex:

female

Basis for effect level:

other: number of implantations; viability index (fetuses)

Dose descriptor:

NOAEL

Effect level:

ca. 2 048.2 mg/kg bw/day

Based on:

test mat.

Remarks:

converted to target substance

Sex:

male

Basis for effect level:

other: pregnancy index

Clinical signs:

not specified

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of viable fetuses decreased dose-dependently

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

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 088 mg/kg bw/day

Based on:

test mat.

Remarks:

converted to target substance

Sex:

male/female

Basis for effect level:

viability

Critical effects observed:

no

Reproductive effects observed:

no

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)

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. 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.

Within this experimental set-up, no relevant adverse effects on reproductive performance at doses of ≥ 352 mg/kg bw/day (P male) and ≥ 358.84 mg/kg bw/day (P female, F) Manganese chloride could be detected. This corresponds to a NOAEL of ≥ 2048 mg/kg bw/d (P male) and ≥ 2088 mg/kg bw/day (P female, F) for the manganese moiety of the target substance. So it can be concluded that the target substance 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. The NOAEL was determined to be ≥ 358.84 mg/kg bw/day. This corresponds to a NOAEL of  ≥ 2048 mg/kg bw/day (P male) and ≥ 2088 mg/kg bw/day (P female, F) for the manganese moiety of the target substance. This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.

Reference 4

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

please refer to table 1

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

please refer to table 1

Organ weight findings including organ / body weight ratios:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Description (incidence and severity):

please refer to table 2

no further details available

Dose descriptor:

NOAEL

Effect level:

ca. 803.2 mg/kg bw/day (nominal)

Based on:

element

Remarks:

converted to target substance

Sex:

female

Basis for effect level:

other: mortality; body weight; haematology (hemoglobin, packed cell volume) number of implantation sites; number of resorptions; pup weight

Critical effects observed:

no

Clinical signs:

not specified

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

please refer to table 3

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings:

not specified

Dose descriptor:

NOAEL

Remarks:

based on intake by dams

Generation:

F1

Effect level:

ca. 803.2 mg/kg bw/day (nominal)

Based on:

element

Remarks:

converted to target substance

Sex:

not specified

Basis for effect level:

other: body weight; dry matter of body weight; ash of dry weight

Reproductive effects observed:

no

Table 1: Body weight gain of female rats during the first eight weeks on diets of different manganese levels, and hemoglobin (Hb) and packed cell volumes values (PCV) of pregnant (21^st) day and non-pregnant rats kept on the different experimental diets (mean ± SE)

Mn mg/kg dry diet	Approx. Mn uptake mg/kg bw/day	Body weight, g Weeks on diet		Hb, g/100 ml		PCV, %
		0	8	Pregnant	Non-pregnant	Pregnant	Non-pregnant
4	0.24	66 ± 1	238 ± 5	9.8 ± 0.5	14.1 ± 0.3	30 ± 2	43 ± 1
24	1.44	66 ± 2	235 ± 7	10.4 ± 0.3	14.3 ± 0.1	34 ± 1	43 ± 1
54	3.24	66 ± 2	235 ± 7	10.2 ± 0.3	14.3 ± 0.2	31 ± 1	43 ± 2
154	9.24	66 ± 1	226 ± 6	9.6 ± 0.6	14.1 ± 0.2	29 ± 2	43 ± 1
504	30.24	66 ± 1	228 ± 6	9.4 ± 0.6	14.0 ± 0.4	30 ± 1	43 ± 1
1004	60.24	66 ± 1	221 ± 5	9.4 ± 0.2	13.6 ± 0.6	30 ± 1	41 ± 2

 

Table 2: Reproductive performance of female rats fed diets of different manganese levels

Mn mg/kg dry diet	Approx. Mn uptake mg/kg bw/day	No of dams	Body weight gain during pregnancy	Implantations per rat	Resorptions	Fetuses
						Dead %	Externally normal per rat
4	0.24	10	117 ± 7	12.6 ± 0.5	10.3	-	11.3 ± 0.7
24	1.44	11	111 ± 6	11.8 ± 1.1	3.8	-	11.4 ± 1.0
54	3.24	11	115 ± 5	12.6 ± 0.8	8.6	0.7	11.5 ± 0.7
154	9.24	10	110 ± 5	11.6 ± 0.7	11.2	-	10.3 ± 0.8
504	30.24	10	99 ± 5	11.9 ± 0.8	12.6	1.7	10.2 ± 0.8
1004	60.24	11	110 ± 6	10.5 ± 0.7	8.7	-	9.5 ± 0.9

 

Table 3: Body weight and body dry matter and ash contents of fetuses from dams fed diets of different manganese levels (mean ± SE)

Mn mg/kg dry diet	Approx. Mn uptake mg/kg bw/day	Body weight g	Dry matter % of bwt.	Ash % of dry wt.
4	0.24	4.63 ± 0.05	13.3 ± 0.1	13.1 ± 0.1
24	1.44	4.72 ± 0.04	13.2 ± 0.1	13.0 ± 0.1
54	3.24	4.73 ± 0.04	13.2 ± 0.1	13.1 ± 0.1
154	9.24	4.67 ± 0.04	13.3 ± 0.1	13.2 ± 0.1
504	30.24	4.38 ± 0.05	13.0 ± 0.1	13.5 ± 0.1
1004	60.24	4.75 ± 0.05	13.0 ± 0.1	13.4 ± 0.1

 

Conclusions:

Although this study was performed according to OECD guideline with deviations, the given data indicate that the study was well-performed, it is consequently reliable with restrictions. 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 803.20 mg/kg bw/day for the manganese moiety of the target substance. Within this experimental set-up, no adverse effects on reproductive performance up to 803.20 mg/kg bw/day (calculated) could be detected and no LOAEL could be determined, so it can be concluded that target substance is not reproductive toxic.

Executive summary:

In a screening reproduction study similar to OECD guideline 421, Manganese sulphate was administered to 17 female Sprague-Dawley rats per dose in diet at dose levels of 4, 24, 54, 154, 504 and 1004 mg/kg feed.

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

There were no compound related adverse effects neither in the dams nor the litters significant compared to control in the main categories systemic or reproductive toxicity. Consequently, no LOAEL could be determined. The NOAEL was determined from the highest dose tested, 1004 mg/kg food. 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 803.20 mg/kg bw/day for the manganese moiety of the target substance (calculated). No LOAEL could be determined, so it can be concluded that the target substance is not reproductive toxic.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

EDDHA

In an one-generation reproduction toxicity study (NOTOX B.V., 1997), the closely related substance Fe(Na)EDDHMA (EC 405 -420 -1) in distilled water was administered to 28 Wistar rats/sex/dose level by single oral gavage (5 mL/kg bw) at dose levels of 50, 200 or 750 mg/kg bw/day (according to the OECD TG 415). A concurrent control group was treated with the vehicle only. Treatment commenced 10 weeks prior to mating for males and 2 weeks prior to mating for females and continued for both sexes until at least the end of the lactation period. Pregnant females were allowed to litter normally. On day 4 of lactation, each litter was adjusted to 4 males and 4 females or as near as possible. The surviving offspring was euthanized as soon as possible after weaning.

The primary effect of treatment with the test item on parental animals was poor physical condition, resulting in premature mortality, growth reduction and reduced food consumption in male and female animals at 750 mg/kg bw/day. These signs of test item-related toxicity were seen with reduced severity at 200 mg/kg bw/day in males only. Thus, the NOAEL for systemic toxicity in parental animals was 50 mg/kg bw/day under the conditions of this study.

In the offspring, increased post natal loss and reduced viability were noted during PND 0 -4 at 200 and 750 mg/kg bw/day, and with lower incidence at 50 mg/kg bw/day. With special regard to the low incidence and unusual distribution pattern of findings noted at 50 mg/kg bw/day, the NOAEL for developmental toxicity was 50 mg/kg bw/day under the conditions of this study.

Based on a slight decrease in the conception indices and a minimal delay in precoital time at 750 mg/kg bw/day, the NOAEL for reproductive performance/fertility was 200 mg/kg bw/day.

Manganese salts

The effect concentrations of manganese compounds were converted to the manganese moiety of the target substance under consideration of the molecular weight and the purity.

 

oral

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.The NOAEL was determined to be ≥ 358.84 mg/kg bw/day.This corresponds to a NOAEL of  ≥ 2048 mg/kg bw/day (P male) and ≥ 2088 mg/kg bw/day (P female, F) for the manganese moiety of the target substance.This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.

 

In a screening reproduction study similar to OECD Guideline 421 (Järvinen et al., 1975), Manganese sulphate was administered to 17 female Sprague-Dawley rats per dose in diet at dose levels of 4, 24, 54, 154, 504 and 1004 mg/kg feed. There were no significant compound related adverse effects neither in the dams nor the litters compared to control in the main categories of systemic or reproductive toxicity. Consequently, no LOAEL could be determined. The NOAEL was determined from the highest dose tested, 1004 mg/kg food, which can be calculated to amount to approx. 60.24 mg/kg bw/day manganese, which corresponds to a NOAEL of 803.20 mg/kg bw/day for the manganese moiety of the target substance. This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.

 

inhalation

In an one-generation reproduction study (equivalent to OECD 415 with deviations) (Dorman et al. 2005), 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. This study is acceptable and satisfies with restrictions the guideline requirement for a one-generation reproductive study (OECD 415) in rats. There were no relevant compound related toxic effects in the main categories of systemic or reproductive toxicity evaluated. No relevant LOAEC could be determined. The NOAEC was derived from the highest dose level tested as ≥ 3.1 mg/m³ air. Converted to the manganese moiety of the target substance, this corresponds to a NOAEC of ≥ 15.03 mg/m³ air.

 

Conclusion

As evident from the results of the available studies, deviations from normal reproductive performance/fertility, as well as impaired developmental and post natal findings in animals treated with manganese salts are not observed. This provides strong evidence that the target substance is not toxic to reproduction. The lowest NOAEL for MnSO₄ derived in a screening reproduction study on rats similar to OECD Guideline 421 (Järvinen et al., 1975), converted to the manganese moiety of the target substance (under consideration of the molecular weight and the purity), was selected as key value for hazard assessment.

Effects on developmental toxicity

Description of key information

There is no data available for the registered substance on developmental toxicity. However, there is data available for the source substances FeNaEDDHA and manganese salts. The values for the manganese salts were converted to the registered substance under consideration of the maximum percentage of manganese included in the substance. This data is used within a frame of a weight-of-evidence approach to assess the toxicity of the target substance. The whole dataset included in the weight-of evidence approach is shown under 'Additional information'. Based on this the key value for manganese was chosen from a reproduction screening study (Järvinen et al. 1975). The NOAEL was determined to be 803.2 mg/kg bw/day for the manganese moiety of the target substance based on the absence of effects in the highest test dose.

In a developmental oral toxicity study (CIBA-GEIGY Limited, 1995) with the source substance Fe(Na)EDDHA in rats, the NOEL for developmental effects was established at the highest dose level of 500 mg/kg bw/day based on the absence of embryo-/foetotoxic or teratogenic effects. Further information can be found under'Additional information'.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Sprague-Dawley derived; Tif:RAIf (SPF); hybrids of RII/1 x RII/2

Clinical signs:

no effects observed

Description (incidence and severity):

There were no treatment-related clinical signs in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no treatment-related deaths in this study. One dam in the mid dose group was sacrificed moribund on day 11 p.c.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in mean maternal body weights between groups at any time during the study.
Maternal body weight gain was statistically significantly reduced in the 500 mg/kg group during the second half of the dosing period (days 11 to 16 p.c.). Body weight gain was unaffected by treatment in the 5 and 100 mg/kg groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg, there was a statistically significant reduction in food consumption during the second half of the dosing period (days 11 to 16 p.c.). At 5 and 100 mg/kg, values for food consumption were similar to those of the control group throughout the study.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Mean carcass weight and gravid uterus weights were not affected by treatment. Net body weight gain was significantly reduced in the 500 mg/kg group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted at necropsy of the dams on day 21 p.c.

Number of abortions:

no effects observed

Description (incidence and severity):

There were no dead or aborted fetuses. The number of live fetuses per litter and fetal weights were not affected by treatment.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Preimplantation losses and early and late postimplantation losses were comparable between groups.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

Preimplantation losses were comparable between groups.

Early or late resorptions:

no effects observed

Description (incidence and severity):

Preimplantation losses were comparable between groups.

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no dead or aborted fetuses. The number of live fetuses per litter and fetal weights were not affected by treatment.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Of the 24 mated animals per group, three, one and three were not pregant in the low, mid and high dose groups, respectively. Thus the number of pregnant animals per group was 24, 21, 23 and 21, respectively. One dam in the mid dose group was sacrificed moribund on day 11 p.c. At necropsy, the number of dams per group with viable fetuses was therefore 24, 21, 22, and 21, respectively.

Details on maternal toxic effects:

There was a reduction in mean food consumption and body weight gain during the second half of the dosing period at 500 mg/kg bw/day.

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Abnormalities:

not specified

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Mean fetal body weights were not affected by treatment.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Fetal sex ratios were not affected by treatment.

External malformations:

no effects observed

Description (incidence and severity):

There were no treatment-related external fetal abnormalities.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related skeletal malformations. Incidental malformations were seen in two control fetuses (no. 13/3: runt, and no. 5/1: umbilical hernia) and one low dose group fetus (no. 33/3: generalized edema, cleft lower jaw and cleft palate). The overall incidence of external, visceral and skeletal anomalies and variations were not affected by treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related findings were observed. Incidental malformations were seen in two control fetuses (no. 13/3: runt, and no. 5/1: umbilical hernia) and one low dose group fetus (no. 33/3: generalized edema, cleft lower jaw and cleft palate). The overall incidence of external, visceral and skeletal anomalies and variations were not affected by treatment.

Details on embryotoxic / teratogenic effects:

There was no indication for test item related embryotoxicity or teratogenic effects under the conditions of this study.

Dose descriptor:

NOEL

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: teratogenicity

Abnormalities:

no effects observed

Developmental effects observed:

no

Chemical Analysis

Administration / Treatment 1:

The mean concentrations of the test item in the vehicle were 88.1 % (0.5 mg/mL) , 94.0 % (10 mg/mL) and 93.3 % (50 mg/mL) of the nominal concentrations for the low mid and high dose groups, respectively. The homogeneity varied in the range from -2 % to 4 % of the mean concentration. The test item was stable in the vehicle.

Administration / Treatment 2:

The mean concentrations of the test item in the vehicle were 94.9 % (0.5 mg/mL) , 93.9 % (10 mg/mL) and 94.1 % (50 mg/mL) of the nominal concentrations for the low mid and high dose groups, respectively. The homogeneity varied in the range from -1 % to 1 % of the mean concentration. The test item was stable in the vehicle.

Conclusions:

Evidence of maternal toxicity, in terms of reduced body weight gain and reduced food consumption, was observed in the high dose group (500 mg/kg). No adverse effects on pregnancy or fetal parameters were observed. On the basis of the results obtained in this study, the no observed effect level (NOEL) was 100 mg/kg body weight/day for the dams and 500 mg/kg body weight/day for the progeny. There was no indication of teratogenic potential. The same result is expected for the target substance since it has the same constituents that act via the same mechanism as the source substance. No differences in the magnitude of the effects are expected. Therefore, the same NOEL is expected also for the target substance.

Executive summary:

In a developmental toxicity study FeNaEDDHA was administered once daily to groups of 24 mated female Sprague-Dawley derived rats by oral gavage at dose levels of 5, 100 or 500 mg/kg bw/day (10 mL/kg bw) from day 6 through day 15 of gestation. Control group females received the vehicle, 0.5 % (w/w) CMC in distilled water, only. The nominal concentrations, homogeneity and stability of the test item in the vehicle were confirmed by chemical analysis of dose formulations. All dams were sacrificed on day 21 of the gestation period and foetuses removed for examination. In dams, there were no treatment-related clinical signs or incidences of mortality. The body weight gain was reduced at 500 mg/kg bw/day for the period of day 6 -16 of gestation, and reduced food consumption was also noted at this dose level. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of teratogenic potential. On the basis of these results, the NOEL was 100 mg/kg bw/day for maternal toxicity and 500 mg/kg bw/day for developmental toxicity and teratogenicity.

The developmental toxicity study in the rat is classified as acceptabel and satisfies the requirements of test guideline OECD 414.

Reference 2

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

CD-1

Clinical signs:

no effects observed

Description (incidence and severity):

The majority of the maternal rats had no observable clinical signs. Clinical signs included alopecia and transient weight loss and were neither clinically relevant nor related to MnSO4 inhalation.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Inhalation exposure to MnSO4 during gestation and lactation did not affect terminal maternal (PND 18) body weight (Table 1).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

MnSO4 exposure was kept constant, no relation to body weight was made.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

no effects (see table 1)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

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.

Dose descriptor:

NOAEC

Remarks:

highest dose tested

Effect level:

15.03 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

Basis for effect level:

body weight and weight gain

clinical signs

organ weights and organ / body weight ratios

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

decreased pup body weights on PND 19

Details on embryotoxic / teratogenic effects:

CLINICAL SIGNS (OFFSPRING)
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)
High-dose manganese exposure (1 mg Mn/m3) was associated with decreased brain weights in PND 14 pups, PND 19 female pups, and PND 45 +- 1 male pups. Analysis of absolute brain weight (relative to body weight) revealed a slight increase in relative brain weights in PND 19 pups occurred. Mean relative brain weights in air-exposed and high-dose MnSO4-exposed PND 19 pups were 3.3 +- 0.09 and 3.9 +- 0.17%, respectively. No gender effect on relative brain weight was observed in the PND 19 pups. Relative brain weights in all other treatment groups were unaffected by MnSO4 exposure. High-dose manganese exposure (1 mg Mn/m3) was also associated with decreased liver weight in PND 19 pups. Male PND 63 pups exposed to MnSO4 at 0.5 mg Mn/m3 had decreased liver weights (13.72 +- 1.07 g) when compared with age-matched air-exposed controls (17.10 +- 0.58 g). All other organ weights measured on PND 63 +-1 were unaffected by MnSO4 exposure (table 2).

Dose descriptor:

NOAEC

Effect level:

15.03 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

Sex:

male/female

Basis for effect level:

other: decreased pup body weights on PND 19, decreased brain weights in some PND 14 to PND 45 pups and decreased liver weight in PND 19 pups. (PND = post natal day)

Dose descriptor:

NOAEC

Effect level:

7.42 mg/m³ air (nominal)

Based on:

test mat.

Remarks:

converted to target substance

Sex:

male/female

Basis for effect level:

other: Organ weights: PND 14: Brain PND 19: Brain, liver PND 45 ± 1: Brain

Abnormalities:

not examined

Developmental effects observed:

no

Calculation of the NOAEC for the target substance:

MW of source substance MnSO4 = 151

MW of Mn = 54.9

Concentration of Mn in target substance : 7.5 %

NOAEC for manganese sulfate =3.1 mg/m³

NOAEC for manganese = 3.1 mg/m³* (54.9/151) = 3.1 mg/m³* 0.36 = 1.13 mg/m³

NOAEC for the target substance = 1.13 mg/m³/ 0.075 = 15.03

calculation method is the same for different values of NOAEC

starting effect concentrations are for following values:

NOAEC: 3.10 mg/m3 air (maternal toxicity: clinical signs, mortality, body weight, organ weights)

NOAEC: 3.10 mg/m3 air (developmental toxicity: clinical signs, mortality, body weight, organ weights)

NOAEC: 1.53 mg/m3 air (organ weights, brain)

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.
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. This corresponds to a value of ≥ 15.03 mg/m³ when converted to the manganese moiety of the target substance.
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 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 corresponds to a value of ≥ 15.03 mg/m³ when converted to the manganese moiety of the target substance.

Reference 3

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Please refer to Read Across Statement attached in Section 13

Reason / purpose for cross-reference:

read-across source

Species:

rat

Strain:

Sprague-Dawley

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

no effects haematology (hemoglobin, packed cell volume) compared to control.

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

not specified

Number of abortions:

not specified

Pre- and post-implantation loss:

not specified

Total litter losses by resorption:

not specified

Early or late resorptions:

not specified

Dead fetuses:

not specified

Changes in pregnancy duration:

not specified

Changes in number of pregnant:

not specified

Other effects:

not specified

Dose descriptor:

NOAEL

Effect level:

ca. 803.2 mg/kg bw/day (nominal)

Based on:

element

Remarks:

converted to target substance

Basis for effect level:

body weight and weight gain

haematology

mortality

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

please refer to table 1

Reduction in number of live offspring:

not specified

Changes in sex ratio:

not specified

Changes in litter size and weights:

not specified

Changes in postnatal survival:

not specified

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Description (incidence and severity):

No gross malformations or bone structure anomalies could be observed in the fetuses.

Visceral malformations:

no effects observed

Description (incidence and severity):

No gross malformations or bone structure anomalies could be observed in the fetuses.

Details on embryotoxic / teratogenic effects:

No gross malformations or bone structure anomalies could be observed in the fetuses, and the manganese intake of the dams was not found to have influenced essentially the fetal weights and the dry matter and ash contents.

Dose descriptor:

NOAEL

Remarks:

based on intake by dams

Effect level:

ca. 803.2 mg/kg bw/day

Based on:

element

Remarks:

converted to target substance

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

Abnormalities:

no effects observed

Developmental effects observed:

no

Table 1: Body weight and body dry matter and ash contents of fetuses from dams fed diets of different manganese levels (mean ± SE)

Mn mg/kg dry diet	Approx. Mn uptake mg/kg bw/day	Body weight g	Dry matter % of bwt.	Ash % of dry wt.
4	0.24	4.63 ± 0.05	13.3 ± 0.1	13.1 ± 0.1
24	1.44	4.72 ± 0.04	13.2 ± 0.1	13.0 ± 0.1
54	3.24	4.73 ± 0.04	13.2 ± 0.1	13.1 ± 0.1
154	9.24	4.67 ± 0.04	13.3 ± 0.1	13.2 ± 0.1
504	30.24	4.38 ± 0.05	13.0 ± 0.1	13.5 ± 0.1
1004	60.24	4.75 ± 0.05	13.0 ± 0.1	13.4 ± 0.1

 

Conclusions:

Although this study was performed according to OECD guideline with deviations, the given data indicate that the study was well-performed, it is consequently reliable with restrictions. 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 803.2 mg/kg bw/day for the manganese moiety of the target substance. Within this experimental set-up, no adverse effects on reproductive performance up to 803.2 mg/kg bw/day (calculated) could be detected.

Executive summary:

In a screening reproduction study similar to OECD guideline 421, Manganese sulphate was administered to 17 female Sprague-Dawley rats per dose in diet at dose levels of 4, 24, 54, 154, 504 and 1004 mg/kg feed. There were no compound related adverse effects neither in the dams nor the litters significant compared to control in the main categories systemic or reproductive toxicity. No gross malformations or bone structure anomalies could be observed in the fetuses, and the manganese intake of the dams was not found to have influenced essentially the fetal weights and the dry matter and ash contents. Consequently, no LOAEL could be determined. The NOAEL was determined from the highest dose tested, 1004 mg/kg food, which corresponds to approx. 60.24 mg/kg bw/day manganese or 803.2 mg/kg bw/day for the manganese moiety of the target substance. This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

EDDHA

In a developmental toxicity study (CIBA-GEIGY, 1995; Report No. 941105), the source substance Fe(Na)EDDHA (CAS 84539 -55 -9) was administered once daily to groups of 24 mated female Sprague-Dawley derived rats by oral gavage at dose levels of 5, 100 or 500 mg/kg bw/day (10 mL/kg bw) from day 6 through day 15 of gestation (according to the OECD Guideline 414). Control group females received the vehicle, 0.5 % (w/w) CMC in distilled water, only. The nominal concentrations, homogeneity and stability of the test item in the vehicle were confirmed by chemical analysis of dose formulations. All dams were sacrificed on day 21 of the gestation period and foetuses removed for examination. In dams, there were no treatment-related clinical signs or incidences of mortality. The body weight gain was reduced at 500 mg/kg bw/day for the period of day 6 -16 of gestation, and reduced food consumption was also noted at this dose level. No adverse effects on pregnancy and no embryo-/foetotoxic effects were observed. There was no indication of teratogenic potential. On the basis of these results, the NOEL was 100 mg/kg bw/day for maternal toxicity and 500 mg/kg bw/day for developmental toxicity and teratogenicity.

 

Manganese salts

The effect concentrations of manganese compounds were converted to the manganese moiety of the target substance under consideration of the molecular weight and the purity.

 

oral

In a screening reproduction study similar to OECD Guideline 421 (Järvinen et al., 1975), Manganese sulphate was administered to 17 female Sprague-Dawley rats per dose in diet at dose levels of 4, 24, 54, 154, 504 and 1004 mg/kg feed. There were no compound related adverse effects neither in the dams nor the litters significant compared to control in the main categories systemic or reproductive toxicity. No gross malformations or bone structure anomalies could be observed in the fetuses, and the manganese intake of the dams was not found to have influenced essentially the fetal weights and the dry matter and ash contents. Consequently, no LOAEL could be determined. The NOAEL was determined from the highest dose tested, 1004 mg/kg food, which corresponds to approx. 60.24 mg/kg bw/day manganese or 803.2 mg/kg bw/day for the manganese moiety of the target substance. This study is acceptable and satisfies with restrictions the guideline requirement for a screening reproductive study (OECD 421) in rats.

 

inhalation

In an one-generation reproduction study (equivalent to OECD 415 with deviations) (Dorman et al., 2005), 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 determined. The NOAEC was derived from the highest dose level tested as ≥3.1 mg/m³ air. Converted to the manganese moiety of the target substance, this corresponds to a NOAEC of ≥15.03 mg/m³ air.

Conclusion

As evident from the results of the available studies, deviations from normal reproductive performance/fertility, as well as impaired developmental and post natal findings in animals treated with manganese compounds are not observed. This provides strong evidence that the target substance is not toxic to reproduction. The lowest NOAEL for MnSO₄ derived in a screening reproduction study on rats similar to OECD Guideline 421 (Järvinen et al., 1975), converted to the manganese moiety of the target substance (under consideration of the molecular weight and the purity), was selected as key value for hazard assessment.

Justification for classification or non-classification

No substance-related effects regarding toxicity to reproduction or developmental toxicity were observed, and so the registered substance does not need to be classified as toxic to reproduction according to Regulation (EC) No 1272/2008.