Manganese

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A GLP and guideline compliant 2 generation reproductive study is available via the relevant inhalation route in rats. Excluding local effects the study the NOAEL for reproductive effects and parental toxicity was considered to be the top dose tested of 20 mg/m3.

Link to relevant study records

Reference

Reference 1

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Study conducted on read-across material

Justification for type of information:

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

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

no guideline followed

Principles of method if other than guideline:

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

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

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


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

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

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

DIET

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

Details on mating procedure:

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

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

Not applicable

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Details on study schedule:

Not reported

Remarks:

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

No. of animals per sex per dose:

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

Control animals:

other: Yes, normal protein diet without Mn exposure

Details on study design:

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

Positive control:

Not reported

Parental animals: Observations and examinations:

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

Oestrous cyclicity (parental animals):

Not reported

Sperm parameters (parental animals):

Not reported

Litter observations:

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


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


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

Postmortem examinations (parental animals):

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

Postmortem examinations (offspring):

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

Statistics:

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

Reproductive indices:

Not reported

Offspring viability indices:

Not reported

Clinical signs:

not specified

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

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


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


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


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

Clinical signs:

effects observed, treatment-related

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

not examined

Histopathological findings:

not examined

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


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


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


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


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


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

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

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

Sex:

not specified

Basis for effect level:

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

Dose descriptor:

conc. level: 3 mg/mL

Generation:

F1

Effect level:

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

Sex:

not specified

Basis for effect level:

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

Reproductive effects observed:

not specified

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

 

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

 

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

 

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

 

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

 

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

 

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

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

Conclusions:

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

Reference 2

Endpoint:

two-generation reproductive toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Reference 3

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2 July 2012 to 4 March 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Remarks:

Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Justification for type of information:

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

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: (F0) 6 - 8 weeks
- Weight at study initiation: (F0) Males: 155 - 298 g; Females: 130 - 194 g
- Housing: Animals were initially housed 2 per cage by sex in polycarbonate cages measuring approximately 61 x 43.5 x 24 cm with stainless steel grid tops and solid bottoms. A few days prior to mating, males were transferred to individual cages with a stainless steel grid insert measuring approximately 48 x 37.5 x 25 cm. After mating, the males were rehoused with their original cage-mates in solid bottomed cages. Mated females were transferred to individual solid bottomed cages (approximately 58.6 x 42.5 x 21 cm). White paper tissues were supplied as nesting material from Day 20 of gestation. Females with litters were retained in this cage type until termination after weaning. F1 animals retained after weaning were housed 2 per cage in cages measuring approximately 61 x 43.5 x 24 cm, as described above. The F1 animals then followed the same caging regime as described for the F0 animals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: F0 animals were acclimatised for 13 days before the commencement of dosing. For at least 7 days prior to commencement of dosing the animals were conditioned to the restraint procedures used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing period of restraint time up to the maximum expected duration to be used on the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 - 26°C
- Humidity (%): 33 - 69%
- Air changes (per hr): at least 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
Test aerosols were generated using a Wright Dust Feed generator device. Exposure of the animals to the test material, or vehicle, was achieved utilising a modular nose only stainless steel flow past inhalation chamber.

- Dose formulation Preparation and analysis
Test material formulation was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh size of 100 μm prior to use, except on one occasion where a sieve mesh of 180 μm was used.

- Preliminary Aerosol Characterisation Investigations
Characterisation of the aerosol generating/exposure system was undertaken prior to commencement of the animal exposures to demonstrate satisfactory performance. Preliminary aerosol characterisation investigations demonstrated that aerosol concentrations were stable spatially within the exposure system and over time and that the particle size distribution investigations showed that test formulation particles for Groups 2 to 4 were respirable for the rat.

- Aerosol Generation
Test item aerosols were generated using a Wright Dust Feed generator device (Wright Dust Feed Mark II, BGI Industries, USA). Prior to the commencement of aerosol generation, a reservoir canister was packed with the test material powder formulation. The powdercake was slowly advanced into the scraper blade at an appropriate speed and scraped powder carried in a pressurised air stream.
The Wright Dust Feed generator device was operated at an appropriate target scraper speed, and air flow rate identified during the preliminary aerosol characterisation investigations. The generated test aerosols were then delivered to the flow past exposure chamber via a connecting tube manifold and mixed with dilution air to achieve the target aerosol concentration. A vacuum pump system was used to continuously exhaust test aerosols from the exposure chamber. Each aerosol generation system was operated to sustain a dynamic airflow sufficient to ensure an evenly distributed exposure aerosol.

- Inhalation Exposure (see Figure 1)
Exposure to the test aerosols was performed using appropriately sized modular nose only stainless steel flow past exposure chamber. Separate inhalation exposure systems were used for the delivery of test aerosol to each treatment group. Each inhalation exposure system was located in an extract booth (to prevent cross-group contamination). This exposure technique allowed a continuous supply of test aerosol to be delivered to each animal; the biased flow created using the flow-past chamber design ensured that there was no re-breathing of the test atmosphere.
For all inhalation exposures, the rats were restrained in clear, tapered, polycarbonate tubes with an adjustable back-stop to prevent the animals from turning in the tubes. The animals’ noses protruded through the anterior end of the restraint tubes which were connected to the exposure chamber by way of a push fit through rubber ‘o’ rings in the chamber wall. This exposure technique was used to minimise concurrent exposure by the oral and dermal routes. The exposure system was operated at an appropriate target total airflow. All flow rates (delivered and extracted) were monitored visually using calibrated flow meters. Exposure chamber flow rates, temperature and relative humidity were monitored and recorded at appropriate intervals during each daily exposure period.


TEST ATMOSPHERE
The aerosol concentration of test material formulation (Groups 2 to 4) or air (Group 1) in the animals’ breathing zone was measured gravimetrically for all groups at regular intervals throughout each daily exposure period.
The test aerosols were sampled using glass-fibre filters (47 mm Whatman GF/B) contained in a stainless steel filter holder in-line with a sampling system comprising a vacuum pump, flow meter and gas meter. Filter samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate to ensure that there was no overloading of the filter which would cause a reduction in sampling flow rate. The filters were weighed before and after sampling and the aerosol concentration calculated using the weight of formulation collected and the volume of air sampled.
In addition to the aerosol chamber concentration assessment, blank filter samples were taken to assess background levels of test material and retained for analysis.
All retained filters from Groups 1 to 4 were placed in amber glass jars and stored in a refrigerator set to maintain 4°C prior to analysis for the determination of the aerosol concentration of test material.
A real time aerosol monitor (Casella Microdust, Casella Measurements, UK) was used to assist in monitoring/ assessing the target concentrations at the start of generation each day and provided a continuous overview of any fluctuations in aerosol concentration.

PARTICLE SIZE DISTRIBUTION
The particle size distribution (PSD) of the test aerosols for Groups 2 to 4 was assessed using a Marple 296 Cascade Impactor. Measurements were undertaken at least once weekly up to Week 8 then at least every 4 weeks thereafter from all groups over the course of the dosing phase of the study. Particle size distribution samples were collected from a reference sampling port representative of the animal exposure ports and test aerosol sampled for an appropriate duration and target flow rate.
The particle size distribution of the test aerosols was determined from the plot of the cumulative percentage (by mass) of particles smaller than the cut-point of each impactor stage against the logarithm of each stage cut-point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis using a computerised linear regression program.

Details on mating procedure:

A few days prior to the initiation of mating, the males were separated into individual grid bottomed cages. Pairings were on a 1 male to 1 female basis. Animals were paired in numerical order within the groups. Each female was transferred to the cage of its appropriate co-group male near the end of the work day, where it remained until mating had occurred or 14 days had elapsed. Vaginal lavages were taken daily early each morning from the day of pairing until mating occurred and the stage of oestrous observed in each lavage recorded. The presence of sperm in such a lavage and/or a copulatory plug in situ was designated as Day 0 of gestation. If the number of males in a group was reduced by mortality, mating was on a 1 male to 2 female basis.
The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The gravimetric filters and particle size distribution samples collected and retained were subjected to chemical analysis using a method validated at Charles River, Edinburgh under Study No. 428133 (Method No. 2813). Full details of the analytical methodology are contained within that report.

Duration of treatment / exposure:

F0 animals were dosed for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation. For F0 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).
From the F1 generation, a group of animals were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation. For F1 males, this treatment continued until the day prior to termination (a total of ca 17 weeks).

Frequency of treatment:

Daily (ca 6 hours per day, 7 days a week)
Females were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of lactation until their litters were born and then exposure was initially reduced to allow the dams to acclimatise to being away from their litter. The females were then dosed as follows:
From Day 1-2 of lactation: ca 1 hour per day
From Day 3-4 of lactation: ca 2 hours per day
From Days 5-20 of lactation until prior to termination (ca Day 21 of lactation): ca 6 hours per day.
Animals that did not litter down, re-commenced/continued dosing until the scheduled termination. Animals that had a litter loss continued on a 6 hour dosing regimen until scheduled sacrifice.

Details on study schedule:

- Selection and Weaning of F1 Animals
From each group, at least 24 males and 24 females were selected for post-weaning assessments. The selected pup(s) were the median’th weight pup(s) of that sex in the litter on Day 21 of lactation. These pups were removed from their mother on Day 21 of lactation, individually identified and housed in a new cage. Pups that were not selected for post-weaning assessments remained with their mother until sacrifice.

Remarks:

Doses / Concentrations:
0, 5, 10, 20 µg/L
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0, 6, 15, 25 µg/L
Basis:
analytical conc.
F0 generation

Remarks:

Doses / Concentrations:
0, 4, 10, 17 µg/L
Basis:
analytical conc.
F1 generation

No. of animals per sex per dose:

- F0 Generation
28 males and 28 females per dose

- F1 Generation
26 animals per sex were dosed at the target concentration of 0 µg/L
24 animals per sex were dosed at the target concentration of 5 µg/L
24 animals per sex were dosed at the target concentration of 10 µg/L
25 animals per sex were dosed at the target concentration of 20 µg/L

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected for use based on results from a preliminary reproduction study in rats (Charles River Study 495849). In addition, guidance values for classification, labelling and packaging (CLP classification) and the inhalable and respirable threshold limit values (TLVs) proposed by the Scientific Committee on Occupational Exposure Limits (SCOEL) were also considered.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- All animals were checked for early each morning and as late as possible each day for viability. Furthermore, all animals were examined for reaction to treatment daily during the course of dosing on the study. The onset, intensity and duration of any signs were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once each week starting in pretrial, all animals received a detailed clinical examination, including appearance, movement and behaviour patterns, skin and hair condition, eyes and mucous membranes, respiration and excreta.

BODY WEIGHT: Yes
- Time schedule for examinations: Weights of F0 animals were recorded one week prior to the first day dosing, then weekly thereafter until the start of the mating period. Males continued to be weighed weekly until termination; but for females, weighing resumed on Day 0 of gestation (the day of detection of
a positive mating sign), and then on Days 7, 14 and 20 of gestation and Days 1, 7, 14 and 21 of lactation (where the day of birth of the litter was designated Day 0 of lactation).
Post-weaning F1 animals were weighed weekly, starting on a suitable day within one week of weaning of the majority of the litters and continued until termination for males and until mating commenced for females. Mated F1 females were weighed on Days 0, 7, 14 and 20 of gestation, then on Days 1, 7, 14 and 21 of lactation. Females that did not show a positive mating sign were weighed weekly until parturition or termination. Females who had a positive mating sign but failed to litter reverted to the weekly weighing regimen following their theoretical Day 24 of gestation.

FOOD CONSUMPTION: Yes
- Time schedule: Food consumption was quantitatively measured for both sexes weekly, starting one week before treatment commenced (F0 animals) or from a suitable day within one week of weaning of the majority of animals (F1 animals) until placement of males in individual cages prior to mating. Weekly measurements continued after the 14 day mating period. For females, following a clear indication of mating, food consumption was measured over Days 0-7, 7-14 and 14-20 of gestation and Days 0-7, 7-14 and 14-21 of lactation

WATER CONSUMPTION: Yes
- Monitoring of water consumption was limited to a visual inspection of the water bottles on a regular basis throughout the study.

OTHERS:
- Observation of Females with Litters during Lactation
The females were allowed to litter normally. If any animal suffered from a difficult or prolonged parturition, this was recorded. The day of birth of the litter (day on which the first pups are born) was designated Day 0 of lactation. The duration of gestation was calculated.
Deficiencies in maternal care were recorded: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

- Seuxal Maturation
Commencing at 28 days of age, females were examined daily for vaginal opening. The day on which the vagina became open was recorded, as was the
body weight on that day. Commencing at 35 days of age, males were examined daily for balano-preputial separation. The day on which separation occurred was recorded, as was the body weight on that day.

Oestrous cyclicity (parental animals):

Over a 2 week period prior to the initiation of mating, vaginal lavages were taken early each morning and the stages of oestrous observed were recorded.

Sperm parameters (parental animals):

The tip of the cauda epididymis was placed in Medium 199 containing 0.2% BSA and HEPES. The sperm were allowed to “swim out” into the medium. An appropriate dilution of the sperm suspension was examined using a Hamilton Thorne sperm motility analyser; sufficient replicates to provide 200 motile sperm were assessed (except where it was obvious that motility was compromised for that animal).
The remaining portion of the cauda epididymis was minced and suspended. Dilutions of this sperm suspension were counted using a haemocytometer to obtain a total sperm count which was expressed per cauda epididymis and per gram of cauda epididymis.
From a sample of the sperm suspension described above, a sperm smear was prepared and stained with eosin. From the Control and High dose animals, two hundred sperm per animal were evaluated for morphological abnormalities using criteria described by Wyrobek and Bruce.
One testis wase decapsulated and homogenized. The homogenate may have been sonicated to remove tissue debris etc, as required. The number of homogenisation resistant spermatids in dilutions of this suspension were counted using a haemocytometer to obtain a total spermatid count which was expressed per testis and per gram of testis.

Litter observations:

The numbers of live and dead pups born in each litter was recorded as soon as possible after completion of parturition on Day 0 of lactation. The live pups were counted and examined from Day 1 onwards for the presence of milk in the stomach and for any externally visible abnormalities daily. The pups were weighed en masse, sexes separated, on Days 1, 4, 7 and 14 of lactation. On Day 21 all pups were weighed individually.
Where practicable, any pups that were found dead or were killed during lactation were sexed and appropriately examined as above. Prior to Day 14 of lactation, any externally abnormal decedent pup was preserved; externally normal ones were discarded. On or after Day 14 of lactation, decedent pups were necropsied.

Postmortem examinations (parental animals):

SACRIFICE
Termination for the adult females was at or shortly after weaning of their litters (Day 21 of lactation). Termination for males was around the time of the termination of the females.
Animals 10 days of age or more were killed by exposure to carbon dioxide followed by exsanguination.

UNSCHEDULED DEATHS
These animals, including those killed or found dead, had a terminal body weight recorded and were necropsied with a view to diagnosis of the cause of the animal’s condition or cause of death. An external examination was followed by inspection of the cranial, thoracic and abdominal contents. The tissues list for animals at scheduled necropsy along with representative samples of abnormal tissues, together with any other tissues considered appropriate, were fixed in neutral 10% formalin. The reproductive tracts of all females were examined for signs of implantation (if they had been paired for mating prior to necropsy), the number of any implantation sites being recorded.

GROSS NECROPSY
Animals were subjected to a complete necropsy examination, which included evaluation of external surfaces and orifices; cranial; thoracic, abdominal, and pelvic cavities with their associated organs and tissues. Necropsy examinations consisted of an external and internal examination and recording of observations for all animals.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

OVARIAN AND UTERINE EXAMINATIONS
The reproductive tract was dissected from the abdominal cavity. The uterus was opened and the contents examined. The reproductive tracts of all females were examined for signs of implantation, the number of any implantation sites being recorded.

HISTOPATHOLOGY
Histological examination was conducted on all adults in the Control and High dose groups of the F0 and F1 generation and a selection of the premature decedents. After a review of the data, histological examination of the respiratory tract tissues of the Control and High dose animals, it was considered appropriate to conduct histopathology on the respiratory tract of all adult animals of the F0 and F1 generation.
The following tissues were processed for microscopic evaluation: adrenal glands, larynx, left testis, left epididymis, lung, bronchial lymph node, cervical lymph node, nasal cavity, ovaries, pharynx, prostate, pituitary gland, seminal vesicles and coagulating glands, trachea (anterior and posterior), uterus (with oviducts and cervix), vagina.
Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment-related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Any cell- or stage-specificity of testicular findings were noted.
The examination of the ovaries included quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.

Postmortem examinations (offspring):

SACRIFICE / GROSS NECROPSY
Pups that were not selected for post-weaning assessments were killed at the same time as their mother.
Animals less than 10 days of age were killed by intra-peritoneal injection of sodium pentobarbitone.

- Offspring found dead or killed (prematurely) before Day 14 of lactation
Where practicable, these animals were sexed, then checked for the presence of milk in the stomach and for the presence of any externally visible abnormalities. Any abnormal pups were, where practicable, fixed in 10% formalin or methylated ethyl alcohol, as appropriate, for optional further examination. Externally normal decedents were discarded.

- Offspring (pre-weaning) found dead or killed (prematurely) on or after Day 14 of lactation
These animals were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin. These carcasses were then discarded.

- F1 and F2 Weanlings at scheduled termination
From each litter, 3 male and 3 female pups (where they were available – if a litter only had females or males, then up to 6 of the relevant sex were selected) were necropsied. This consisted of an external examination followed by macroscopic examination of the tissues and organs of the cranial, thoracic and abdominal cavities in situ. Samples of any grossly abnormal tissues were preserved in 10% formalin. From one of the 3 pups of each sex, the weights of the brain, spleen and thymus were recorded, and these organs were preserved. Representative samples of any abnormal tissues from any of the 6 pups were also
preserved. The carcasses were then discarded.
The remaining pups in each litter were checked for externally visible abnormalities at the time of killing. Any found to have such an abnormality were necropsied as described in the preceding paragraph. The remaining carcasses were discarded.

ORGAN WEIGHTS
The following were weighed: brain, epididymides, adrenal glands, pituitary gland, prostate glang, thyroid glands, kidneys, liver, lung, ovaries, spleen, testes, uterus.

HISTOPATHOLOGY
Histological examination was conducted on the brain, spleen and thymus of Control and High dose F1 and F2 weanlings (the selected weanlings at necropsy). A single H&E section was cut, stained and evaluated.

Statistics:

Unless otherwise stated, all statistical tests were two-sided and performed at the 5% significance level using in house software. Pairwise comparisons were only performed against the control group.
Select body weight and food consumption were analysed for homogeneity of variance using the ‘F-Max’ test. If the group variance appeared homogeneous, a parametric ANOVA was used and pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test ie pairwise comparisons was made only if the overall F-test was significant. If the variances were heterogeneous, log or square root transformations were used in an attempt to stabilize the variances. If the variances remained heterogeneous, then a Kruskal-Wallis non-parametric ANOVA was used and pairwise comparisons were made using chi squared protection (Via z tests, the non-parametric equivalent of Student’s t test).
Organ weight data was analysed as above, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Reproductive indices:

For each group the following were calculated:

Fertility Index (male) = number siring a litter / Number paired

Fertility Index (female) = Number pregnant / Number paired

Gestation Index = Number bearing live pups / Number pregnant

Offspring viability indices:

For each litter and group the following were calculated:

Birth Index = Total number of pups born (alive and dead) / Number of implantation scars

Live Birth Index = Total number of pups live on Day 0 of lactation / Total number born (live and dead)

Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0

Lactation Index = Number of pups live on Day 21 of lactation / Number live on Day 4

Overall Survival Index = Number of pups live on Day 21 of lactation / Total number born (live and dead)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

See 'Details on effects (parental animals)' for further information

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

See 'Details on effects (parental animals)' for further information

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

See 'Details on effects (parental animals)' for further information

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

See 'Details on effects (parental animals)' for further information

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

MORTALITY (PARENTAL ANIMALS)
- F0 animals
Animal 138 (Group 1F) was killed prematurely on Day 97 of the study. The animal was sacrificed at the time of parturition as the animal had difficulty giving birth and there was a pup protruding from the vagina (the animal gave birth to one live pup). The uterus also contained live foetuses and one late death. Animal 330 (Group 3F) was killed prematurely on Day 94 of the study. The animal had a prolonged parturition and had given birth to 3 live pups. One dead foetus was found in the right uterine horn at necropsy. There were no abnormalities detected at histological evaluation.
Animals 228 (Group 2M) and 236 (Group 2F) were killed prematurely on Day 85 and Day 83, respectively due to clinical signs. The male animal had shavings stained red, a cold body, reduced activity, rolling gait, staggering and weight loss. Necropsy findings for this animal included yellow froth filled duodenum, ileum and jejenum, pale foci on kidney, pale foci left lung lobe, enlargement of adrenal gland, small thymus, urinary bladder adhesions. Histological findings included a mild ulcer in the larynx. The female had partially closed eyes, dilated pupils, tremors, unkempt coat, walking on tip toes, irregular respiration, staggering and subdued. Necropsy findings included pale extremities and fluid accumulation in both horns of the uterus (the animal was sacrificed prior to having a clear indication of mating). There were no abnormalities detected at histological evaluation.
There was no treatment related pattern to these deaths and these were not positively attributed to treatment.
- F1 animals
Animal 521 (Group 1M), animal 717 (Group 3M), animal 748 (Group 3F), Animal 751 (Group 3F) and animal 816 (Group 4M) were killed prematurely. However, none of these premature deaths were considered to be related to treatment but were considered to be due to accidental injury.

CLINICAL SIGNS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there were 2/28 males noted as having wheezing respiration. Animal 333 (Group 3F) had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over ca Days 83-90 of the study. Due to the signs dosing for the animal was stopped for a few days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals, these signs were considered to be incidental. Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
- F1 animals
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).

BODY WEIGHT (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was a decrease in body weight gain in males over Days 0-21 of the study. From Day 21 of the study, the body weight gains were generally comparable to the controls but the group mean weights remained lower than the controls throughout the study. At target 20 μg/L, there was a group mean body weight gain in females prior to mating were similar to the controls, however body weight gains over Days 0-20 of gestation were slightly lower than the controls. Gains over lactation were similar to the controls.
- F1 animals
At target 20 μg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study, however gains over the following week were greater than the controls and then remained comparable with the controls throughout the remainder of the treatment period. Slight intergroup differences in group mean body weight gains in the F1 females prior to mating were too small to be attributed to treatment. At 20 μg/L, there was a slight reduction in body weight gains throughout gestation compared to the controls.
There were no effects of treatment noted in the lactation females.

FOOD CONSUMPTION (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls. At target 20 μg/L, there was a transient reduction in food consumption in the females on commencement of treatment compared with the controls; however, consumption for the remainder of the pre-mating period was similar to the controls. Slight intergroup differences in the group mean food consumption in the males at target 5 μg/L and target 10 μg/L were not attributed to treatment. Slight intergroup differences in group mean food consumption throughout gestation and lactation were not attributed to treatment.
- F1 animals
At target 20 μg/L, there was a slight reduction in group mean food consumption in the males over Days 40-68 of the study; these reductions achieved statistical significance. Slight intergroup differences in group mean food consumption at target 5 μg/L and target 10 μg/L were not attributed to treatment. Group mean food consumption in the females prior to mating and throughout gestation and lactation were comparable to the controls.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The stages of the oestrus cycles and their mean duration were similar in all groups for both generations.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no effects on the sperm motility, count or morphology at any of the dose levels applied, in either generation.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no effects of treatment on mating performance, fertility or duration of gestation in either generation.

ORGAN WEIGHTS (PARENTAL ANIMALS)
- F0 animals
At target 20 μg/L, reduced brain weights in males achieved statistical significance (P<0.05) compared with controls. However, the lower body weight was also statistically significant (P<0.05) following covariance analysis brain weight did not achieve significance and therefore was not positively attributed to treatment. In all treated females, there was a statistically significant increase in lung weights, compared with the controls; these increases were still present following covariance analysis (P<0.01 at target 5 μg/L and P<0.001 at target 10 and 20 μg/L). Other slight differences in organ weights such as an increased thyroid weight in males at target 5 μg/L and an increase in kidney weights of females at target 10 μg/L were not attributed
to treatment.
- F1 animals
At target 5 and 10 μg/L, kidney weights in males were statistically higher than the control, however there was no dose relationship to this increase and following covariance analysis, these findings were no longer evident. At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights in females (P<0.05 at target 10 μg/L and P<0.001 at target 20 μg/L) following covariance analysis. Other slight differences in organ weights such as an increased adrenal weight in females at target 20 μg/L were not attributed to treatment.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to administration ofthe test material.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no treatment related findings observed in the reproductive tract in the F0 or F1 generations.
Histological findings were confined to the respiratory tract. Inhalation of the test material was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.

OTHER FINDINGS (PARENTAL ANIMALS)
- Sexual Maturation
The age and body weight at preputial separation or vaginal opening of the F1 generation animals in all treated groups was similar to the controls.

Dose descriptor:

NOEL

Effect level:

20 mg/m³ air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment related effects were observed

Remarks on result:

other: Generation: F0 and F1

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

LITER SIZE AND PUP MORTALITY
- F0 generation, F1 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 20 μg/L, there was an increase in the number of animals losing more than 2 pups at birth (total pups born/no. of implantation sites). However, the mean birth index (%) was well within the background range and these increases were considered to be incidental.
- F1 generation, F2 production
The mean number of implant sites and total number of pups born in all groups was comparable to controls.
At target 10 and 20 μg/L, pup survival (no. losing >3 pups) over Days 0-4 of lactation was slightly lower than the controls. However, the number of animals losing the entire litter was comparable with controls and the remaining animals generally lost 4 pups. In addition, there was no clear dose related response to these reductions and these were considered not to be an effect of treatment.

LITTER AND PUP WEIGHTS
- F0 Generation
In all treated groups, group mean litter and pup weights were comparable to the controls.
- F1 Generation
At target 20 μg/L, group mean litter weights were slightly lower than the controls which reflected the smaller litter size at this level. However, although the litter weights were slightly lower than the controls, the mean pup weights in both males and females were comparable to the controls.

ORGAN WEIGHTS
- F0 generation, F1 production
At target 20 μg/L, there was a reduction in thymus weight of the females, compared with the controls (P<0.01). Following covariance analysis, this reduction did not achieve statistical significance. There were no effects on organ weights at target 5 and 10 μg/L.
- F1 generation, F2 production
Slight intergroup differences in organ weights did not achieve statistical significance and were attributed to treatment.

GROSS PATHOLOGY
There were no treatment related gross findings recorded. The findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence in control and treated animals and, therefore, were considered unrelated to treatment with the test material.

HISTOPATHOLOGY
There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.

Reproductive effects observed:

not specified

Blood Analysis Results

F0 Males
Time-point	Blood Mn conc (ppb w/v (ng/mL))
	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	7	7	7	6
Prior to mating	6	13	23	27
Prior to Necropsy	6	19	27	29
F0 Females
Time-point	Blood Mn conc (ppb w/v (ng/mL))
	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	7	7	7	7
Prior to mating	6	16	28	39
Prior to Necropsy	7	16	24	33

At target 20 μg/L, manganese levels prior to mating were 350% higher than controls in males and 550% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 383% and 371% for males and females.

At target 10 μg/L, manganese levels prior to mating were 283% higher than controls in males and 367% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 350% and 243% for males and females.

At target 5 μg/L, manganese levels prior to mating were 117% higher than controls in males and 167% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 217% and 129% for males and females.

F1 Males
Time-point	Blood Mn conc (ppb w/v (ng/mL))
	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	12	16	16	17
Prior to mating	6	9	13	19
Prior to Necropsy	6	9	14	21
F1 Females
Time-point	Blood Mn conc (ppb w/v (ng/mL))
	Group 1 (Control)	Group 2 (5 µg/L)	Group 3 (10 µg/L)	Group 4 (20 µg/L)
Pre-treatment	13	12	15	15
Prior to mating	6	10	16	23
Prior to Necropsy	7	10	16	21

At target 20 μg/L, manganese levels prior to mating were 217% higher than controls in males and 283% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 250% and 200% for males and females.

At target 10 μg/L, manganese levels prior to mating were 112% higher than controls in males and 133% higher than controls in females at the pre-mating timepoint. At terminal necropsy, these values were 167% and 129% for males and females.

At target 5 μg/L, manganese levels prior to mating were 50% higher than controls in males and females at the pre-mating timepoint. At terminal necropsy, these values were 50% and 43% for males and females.

The manganese concentrations in the blood of all the treated F1 animals were lower than the same time-point levels of the F0 generation animals.

Conclusions:

Under the conditions of the study the No Observed Effect Level (NOEL) of manganese chloride, for reproductive toxicity, was determined to be 20 µg/L.

Executive summary:

The reproductive toxicity of manganese chloride was investigated in a two generation study which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800.

F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed with manganese chrloride for 10 weeks prior to mating, and then throughout mating, gestation and lactation until termination after the F1 generation had reached Day 21 of lactation.

From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on study and were dosed for approximately 11 weeks after weaning, and then throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.

Animals were monitored for clinical signs of toxicity and for effects on body weight, food consumption, effects on oestrous cycles, mating performance, pregnancy performance, difficulty or prolongation of parturition, and for deficiencies in maternal care. The offspring were monitored for survival and growth up to weaning. In addition, the following endpoints were evaluated: gross necropsy findings, organ weights, histopathology evaluation, qualitative examination of testes and examination of the ovaries and sperm evaluation. Blood samples were taken from all adult animals for bioanalytical analysis prior to dosing, prior to mating and prior to weaning/necropsy.

Clinical signs of reaction to treatment to inhalation exposure of manganese chloride were confined to a few animals with wheezing respiration in the F0 generation exposed to target levels of 10 and 20 μg/L.At target 20 μg/L, overall body weights and food consumption of the F0 males throughout the study were lower than controls. In the F1 generation, the body weight gain of the males at target 20 μg/L were transiently reduced on commencement of treatment; in addition, the food consumption at this level was lower than the controls over Days 19-68 of treatment. At target 20 μg/L, there was a slight reduction in group mean body weight gains during gestation in both generations. Gains throughout lactation were similar to controls.

There was no effect of treatment on oestrous cycles, mating performance, fertility or duration of gestation or litter size in either generation. Slight intergroup differences in the pup survival were too small to be attributed to treatment. Group mean litter and pup weights in the F0 generation litters were comparable with controls. At target 20 μg/L, group mean litter weights were slightly lower than the controls, however this reflected a slightly smaller litter size at this level. The mean pup weights in both males and females were comparable to the controls and the slightly lower litter weights were not attributed to treatment.There were no effects of treatment on the sexual maturity of the F1 animals.

At target 10 and 20 μg/L, there was a statistically significant increase in kidney weights compared to the controls, however there was no alteration in the normal structure of these organs, as seen by microscopy (at target 20 μg/L).In all treated F0 females, there was a statistically significant increase in lung weights compared to the controls; this increase in lung weights was not evident in the F1 females.

There was no effect of treatment on the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation.

Inhalation of manganese chloride was associated with microscopic findings in the nasal cavity, larynx, lung and trachea (including carina) in all dose groups of the F0 generation, in the pharynx of F0 generation animals exposed to target 10 and 20 μg/L; in the nasal cavity, pharynx, larynx and lung in all dosed group of the F1 generation and in the trachea (including carina) of F1 generation animals exposed to target 10 and 20 μg/L.No test substance-related findings were observed in the reproductive tract in the F0 or F1 generations and in tissues examined from weanlings in the F1 and F2 generations.

In all treated groups of the F0 generation, the levels of manganese in the blood increased significantly on commencement of dosing (as recorded prior to mating) in both males and females. The concentrations recorded prior to mating and prior to necropsy were comparable in all groups which did not indicate any obvious accumulation over the dosing period. In the F1 generation, pre-treatment concentrations in all groups were higher than the F0 generation pre-treatment values. In addition, at target 5 and 10 μg/L in the F1 generation, the pre-treatment values were generally higher or similar to the values recorded during the dosing period, indicating that the exposure to the test substance through the mother’s milk during lactation resulted in an increased exposure to the test substance in the F1 animals from birth. At target 20 μg/L, the concentrations of the F1 males and females throughout the dosing period were greater than the pre-treatment values indicating an increased exposure throughout the dosing period.

In conclusion, under the conditions of this study, a No Observed Effect Level for adult effects was not established due to effects on the respiratory tract. The No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level 20 μg/L.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

325 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

This should be considered a limited supporting study, which indicated no reproductive, lactational or offspring effects. Oestrus cyclicity and sperm parameters not measured.

Effect on fertility: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

20 mg/m³

Study duration:

chronic

Species:

rat

Quality of whole database:

Data point addressed by recommended guideline and GLP compliant study.

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In accordance with REACH Annex XI, Section 1.1; use of existing data, reproductive toxicity testing is not considered to be required since data are available on a more soluble (and therefore more bioavailable) Mn substance, manganese chloride (MnCl2). Because the bioavailability of manganese ions from manganese chloride would be substantially greater, the results can be considered a worst-case for manganese. An OECD 416 study has been completed for manganese chloride in the rat (Jardine, 2013). Under the conditions of this study, there were no adverse effects on the reproductive performance of the animals up to the target dose level - 20 µg/L.

Furthermore, protecting for neurotoxicity (STOT RE), which is considered a more sensitive endpoint for manganese substances than reproductive toxicity, would by default protect for reproductive toxicity. In addition, not a single available literature source from a literature review dating back 50 years (on human and animal data on reproductive toxicity (all aspects) to manganese-based compounds) hints or suggests that Mn specifically could cause reproductive toxicity. Being an essential nutrient, necessary for the formation of bones, coupled with its poor absorption and the efficient homeostatic control of manganese in the body, it is very unlikely that Mn will cause reproductive effects. This also can be supported by the absence reprotoxicity reported in Jardine (2013).

Testing is therefore considered unlikely to provide any additional or useful information and is also considered unjustified on animal welfare grounds, particularly considering the high number of animals that would be required.

The inhalative reproductive toxicity of the test material was investigated in a two generation study (Jardine, 2015) which was conducted under GLP conditions and in accordance with the standardised guidelines OECD 416 and EPA OPPTS 870.3800. Male and female Sprague-Dawley rats were exposed to the test material via the inhalation route at target concentrations of 0, 5, 10 and 20 µg/L. Under the conditions of this study, a No Observed Effect Level (NOEL) for adult effects was not established due to effects on the respiratory tract. However, the respiratory tract effects observed are commonly observed in irritant materials and were considered not to be a unique effect of the test material. Under the conditions of the study the No Observed Adverse Effect Level (NOAEL) for the parental animals was determined to be 20 µg/L. The NOEL for reproductive toxicity was determined to be 20 µg/L.

In an oral study (Ali et al, 1983 ), Mn exposure had no significant effect on growth pattern, brain weight or brain and plasma protein contents at 325 mg/kg bw/day. Diet regimen had no effect on accumulation of Mn in any group but levels were higher in F1 pups. In F1 pups Mn exposure had no effect on eye opening in either group, delayed startle reflex in low protein group only but air righting reflex development delayed in both dietary groups, more marked in low protein group.

Short description of key information:
A GLP and guideline compliant 2 generation reproductive study is available via the relevant inhalation route in rats. Excluding local effects the study the NOAEL for reproductive effects and parental toxicity was considered to be the top dose tested of 20 mg/m3.

Justification for selection of Effect on fertility via oral route:
Non guideline compliant one generation study on MnCl2. Best study available via the oral route.

Justification for selection of Effect on fertility via inhalation route:
Guideline and GLP compliant study via the inhalation route.

Effects on developmental toxicity

Description of key information

Good developmental toxicity data (OECD 414 compliant or equivalent) exists in two species (rat and mouse) by the inhalation (rat) and dermal (mouse) routes - both of which are considered to bypass the liver, and hence are both considered relevant to inhalation exposure of MnCl2.  NOAELs were identified  in both studies and identified similar developmental effects.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 January 2014 to 30 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Remarks:

Study conducted on read-across material

Justification for type of information:

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

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines (MAFF, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Teratology (2-1-18), Agricultural Production Bureau, dated November 24, 2000)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: RccHan™: WIST(SPF)
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation: 203 to 262 g (Day 0 post coitum)
- Housing: Group A females (mated) were housed in groups of three to five animals in cages with wire mesh tops up to the day of mating and afterwards individually in cages with wire mesh tops. Group B females (not mated) were housed individually in cages with wire mesh tops. Cages were equipped with sterilised standard softwood bedding with paper enrichment.
- Diet: Pelleted standard rodent maintenance diet (ad libitum)
- Water: Community tap water in water bottles (ad libitum)
- Acclimation period: Animals were acclimated under test conditions after a health examination. Dams were accustomed to the restraining tubes for 3 daily periods of approximately 1, 2, and 4 hours, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 % (relative)
- Air changes: 10 - 15 air changes per hour
- Photoperiod: There was a 12-hour fluorescent light / 12-hour darkness cycle with music during the light period.

IN-LIFE DATES:
From: 28 Jan 2014
To: 28 April 2014

Route of administration:

inhalation: aerosol

Type of inhalation exposure (if applicable):

nose only

Vehicle:

air

Details on exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past system. Ports for animal exposure were positioned radially around the nose-only, flow-past exposure chamber on several different levels. The aerosol was discharged constantly through the exposure system. The exposure system ensured a uniform distribution and provided a constant flow of test material to each exposure tube. Before commencement of the exposure of the group(s), technical trials were conducted (without animals) using the inhalation system foreseen for the study.
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes.
- System of generating particulates/aerosols: A dust aerosol was generated from the test material using a rotating brush aerosol generator connected to a micronising jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutraliser. Furthermore, the aerosol concentrations of the test material of the low dose group were achieved by serial dilution with compressed, filtered, dry air of the higher aerosol concentration of the mid dose group using an air vacuum device.
- Temperature, humidity, pressure in air chamber: Aerosol concentration, particle size distribution, relative humidity and temperature were measured on test aerosol samples taken at a representative exposure port. The relative humidity and temperature in the chamber were measured continuously during each exposure using a calibrated device. Additionally, values were recorded hourly during each exposure.
- Oxygen concentration: The oxygen concentration was measured on test aerosol samples taken at a representative exposure port. The oxygen concentration in the chamber was measured during each exposure using a calibrated device. Additionally, values were recorded hourly by hand during each exposure. The oxygen concentration was maintained above 19 % during the exposure period.
- Air flow rate: The flow of air at each tube was 1 L/min, which is sufficient to minimise re-breathing of the test aerosol as it is more than twice the respiratory minute volume of a rat. All airflow rates (including those for concentration and particle size measurements) were determined using calibrated gas meters and pressure gauges or flow meters. The exposure airflow rate was adjusted as appropriate before the start of the exposure using calibrated flow-meters and/or pressure gauges. The actual airflow rate was monitored hourly during each exposure. Additional measurements were performed if considered necessary.
- Method of particle size determination: The particle size distribution was determined gravimetrically three times for the low, mid and high dose groups. The cumulative particle size distribution of the test aerosol was determined using a Mercer 7 stage cascade impactor Model 02-130 (In-Tox. Products Inc., Albuquerque, New Mexico, USA). The test aerosol was impacted at each stage onto stainless steel slips and the particle size distribution of the test material in the generated aerosol was measured by gravimetrically analysing the test material deposited on each stage of the cascade impactor. The airflow rate through the impactor was 1 L/min. The mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD) were calculated on the basis of the gravimetric results from the impactor, using Microsoft Excel® software. The target ranges were 1 to 3 μm for the MMAD and 1.5 to 3 for the GSD.
- Treatment of exhaust air: The aerosol was exhausted using a tubing/filter system.

TEST ATMOSPHERE
- Brief description of analytical method used:

>Determination of Nominal Aerosol Concentration
The test material usage was measured during each exposure in the mid and high dose groups by weighing the generator cylinders containing the test material before and after each exposure to determine the quantity of test material used. The weight used was then divided by the total air-flow volume to give the nominal concentration. The nominal concentration of the low dose group was calculated from the value of the mid dose group under consideration of the dilution factor. These data were used for the purpose of monitoring the performance of the generation system.

>Gravimetric Determination of Aerosol Concentration
Gravimetric determination of the aerosol concentration was performed twice to four times per exposure for the low, mid and high dose groups. Additional samples were collected for monitoring purposes.

VEHICLE
- Composition of vehicle: Compressed, filtered, dry air

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test aerosol samples were collected onto Millipore® durapore filters, type HVLP using a stainless steel filter sampling device. Sampling flow was similar to the air flow rate per exposure port. The filters were weighed before and at least 10 minutes after sampling using a calibrated balance. The gravimetric aerosol concentration was calculated from the amount of test material present on the filter and the sample volume. A correction factor of 1.67 (determined from the technical trials) was applied to correct for the adsorption of water during sampling due to the hygroscopic properties of the test material. This factor was determined during technical trials by AAS analysis on the Mn content and was confirmed by additional AAS analysis of filters taken during exposure. For AAS analysis filter samples were sent to the person responsible for dose formulation analysis.

FORMULATION ANALYSIS
- Analytical Standard
Manganese 1000 μg/mL AAS/ICP

- Study Samples and Storage
Filter samples were dispatched to the analytical laboratories internally (at room temperature) and directly analysed.

- Purified water
Prepared in-house with an ELGA water purification system (Ultra Bio No. UBH 279651)

ANALYTICAL PROCEDURE
- Preparation of Calibration Solutions
A stock solution of analytical standard in 1 M chloride acid (HCl) with a concentration of 2.56 μg/mL was prepared (solution A) by dissolving 256 μL of the analytical standard in 100 mL of 1 M chloride acid. Standard solutions were prepared by successive dilution of solution A with 1 M chloride acid. The resulting concentrations ranged from 0.040 to 1.280 μg/mL. These standard solutions as well as solution A were used to calibrate the atomic absorption spectrometer.

- Work up of Samples
An appropriate volume of 1 M chloride acid was added to each filter sample and dissolution was achieved by sonication for at least 5 minutes.

-Atomic Absorption Spectrometry with Flame Assembly
Instrument: Perkin-Elmer Model PE 2100 (software 4100) atomic absorption spectrometer
Flame: Acetylene flame/air
Slit Width: 0.2
Wavelength: Calcium: 279.5 nm

- Evaluation of Results
Samples were quantified by atomic absorption spectrometry (AAS) of manganese with reference to the respective calibration curve (with zero intercept). The calibration curve (non-linear) and the concentration (in μg/mL) were calculated using the Perkin Elmer software.
The concentration of precipitated test material in the filter samples was calculated using the following equation:
Filters: A(filter) = (Cs ∙ V ∙ D ∙ F) / 1000
where
A(filter) = Actual amount of test material on filter [μg/filter]
Cs = Measured concentration of manganese in sample [μg/mL]
V = Volume solvent for dissolution [mL]
D = Dilution factor
F = Correction factor of 2.2906

Details on mating procedure:

- Impregnation procedure: Cohoused. After acclimatisation, females were housed with sexually mature males in special automatic mating cages i.e. with synchronised timing to initiate the nightly mating period, until evidence of copulation was observed. This system reduced the variation in the copulation times of the different females.
- M/F ratio per cage: 1:1
- Length of cohabitation: Not reported
- Proof of pregnancy: The females were removed and housed individually if the daily vaginal smear was sperm positive or a copulation plug was observed. The day of mating was designated day 0 post coitum.
- Other: Male rats of the same source and strain were used only for mating. These male rats are in the possession of laboratory and were not considered part of the test system. The fertility of these males had been proven and was continuously monitored. Females in recovery groups were not mated.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

Animals were treated with the test material once daily at approximately 24 hour intervals.

Duration of test:

Females were treated for 15 consecutive days. Mated females were treated from days 6 to 20 post coitum) and recovery animals from day 1 to 15 of a concurrent treatment period.
The recovery period was 8 weeks.

No. of animals per sex per dose:

Females A: 88 mated females, 22 per group
Females B: 24 not mated females, 6 per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous developmental neurotoxicity study in Han Wistar rats conducted at the testing facility using aerosol concentrations of 5, 15 and 25 µg/L air. At a dose level of 25 µg/L laboured breathing and reduced body weight were observed in dams after treatment during gestation. No test material-related effects were recorded in breeding at any aerosol concentration for the test material.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed for viability/mortality twice daily. Daily cage-side clinical observations were made once daily during acclimatisation and after treatment up to the day of necropsy.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: For Group A, body weights were recorded daily from day 0 until day 21 post coitum. For Group B, body weights were recorded on treatment days 1, 8 and 15 and recovery days 1, 8, 15, 22, 29, 36, 43, 50 and 57.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: For Group A, food consumption was recorded at 3-day intervals on days 0 - 3, 3 - 6, 6 - 9, 9 - 12, 12 - 15, 15 - 18 and 18 - 21 post coitum.
For Group B, food consumption was recorded on treatment days 1 - 8 and 8 – 15 and recovery days 1 - 8, 8 - 15, 15 - 22, 22 - 29, 29 - 36, 36 - 43, 43 – 50 and 50 – 57.

WATER CONSUMPTION AND COMPOUND INTAKE: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice
At the scheduled necropsy on day 21 post coitum, main study females were sacrificed by CO₂ asphyxiation and the foetuses were removed by Caesarean section. Recovery females were sacrificed by intraperitoneal injection of pentobarbitone after 4 (3 females per group) or 8 weeks (3 females per group) of recovery period.

-Necropsy
Group A: Any female sacrificed during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs was performed. The uteri (and contents) of all females with live foetuses were weighed during necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain.
When considered appropriate, macroscopic changes in the dams were photographed and samples of tissue fixed in neutral phosphate buffered 4 % formaldehyde solution for possible microscopic examination.
One foetus from 6 different litters of each dose group was removed, weighed and stored at -20 ± 5 °C for possible determination of test material levels. In agreement with the Sponsor, these foetuses were discarded after delivery of the draft report.

- Tissue Preservation
At scheduled sacrifice, the lungs from certain females were preserved; the lungs from 6 pregnant females per dose group, all non-pregnant females per dose group and all 6 recovery females were preserved in neutral phosphate buffered 4 % formaldehyde solution.

-Histotechnique
The lungs from pregnant Group A females in the control and high-dose group as well as all occurring gross lesions were processed, embedded and cut at an approximate thickness of 4 micrometres and stained with haematoxylin and eosin.
Treatment-related changes were observed in the lungs of pregnant females at the high-dose, therefore the lungs of pregnant females in groups 2 and 3 and all recovery females were processed.

- Histopathology
Slides of all organs and tissues collected at terminal sacrifice of the control and high-dose group were examined.
Test material-related morphologic changes were detected in organs of high-dose animals and therefore the lungs from the remaining groups were examined to establish a no-effect level, if possible.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Group A: Any female sacrificed during the study was subjected to macroscopic examination with emphasis on the uterus and its contents. Post mortem examination, including gross macroscopic examination of all internal organs with emphasis on the uterus, uterine contents, corpora lutea count and position of foetuses in the uterus was performed.
If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible haemorrhagic areas of implantation sites.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: half per litter

Foetuses were removed from the uterus, sexed, weighed individually, examined for gross external abnormalities, sacrificed by a subcutaneous injection of sodium pentobarbital and allocated to one of the following procedures:
- Microdissection technique (sectioning/dissection technique). At least one half of the foetuses from each litter was fixed in Bouin's fixative (one foetus per container). They were examined by a combination of serial sections of the head and microdissection of the thorax and abdomen. This included detailed examination of the major blood vessels and sectioning of the heart and kidneys. After examination, the tissue was preserved in a solution of glycerin/ethanol (one foetus per container). Descriptions of any abnormalities and variations were recorded.
- The remaining foetuses were eviscerated and with the exception of over the paws, the skin was removed and discarded. Carcasses were processed through solutions of ethanol, glacial acetic acid with Alcian blue (for cartilage staining), potassium hydroxide with Alizarin red S (for clearing and staining ossified bone) and aqueous glycerin for preservation and storage. The skeletons were examined and all abnormal findings and variations were recorded. The specimens were preserved individually in plastic vials. The assessment included, but was not limited to all principal skeletal structures including cranium, vertebral column, rib cage and sternum, pectoral and pelvic girdles. After the staining of the foetuses for skeletal examination, specimens were evaluated. Foetuses were examined in mixed group order. Each litter was examined in sequential order. Foetuses with abnormalities were photographed when considered appropriate.

- Histotechnique and Histopathology
An increased number of large thyroids was found in group 4 during visceral examination of foetuses and therefore this organ was examined histopathologically to establish whether the increase in size is related to any microscopic change.
To this purpose, normal thyroids from ten foetuses in the control group and thyroids with increased size from ten foetuses in the high-dose group were selected as follows:
- in the control group one foetus per litter were randomly selected to represent ten litters;
- in the high-dose group, all five foetuses (from four litters) with large thyroid were selected and additionally five foetuses with slightly large thyroid were selected from five different litters.
Foetal thyroids were trimmed transversely leaving them attached to the trachea. They were embedded on this cut surface and serial section were cut at 4 μm. They were then stained with haematoxylin and eosin.

Statistics:

The following statistical methods were used to analyse food consumption, body weights, reproduction and skeletal examination data:
- Means and standard deviations of various data were calculated and included in the report.
- The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
- Fisher's exact-test was applied if the variables could be dichotomised without loss of information.

The skeletal examination data were first assessed using Bartlett’s test for homogeneity of variance. As these data were found to be non-homogenous, non-parametric assessment by Kruskall-Wallis and, if significant, pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test was used.

Historical control data:

Historical control data were included in the report.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
MORTALITY AND CLINICAL SIGNS
GROUP A
All females survived the scheduled study period.
Treatment with the test material caused breathing noises and dyspnea in females in groups 3 and 4. In group 4, breathing noises were observed on day 8 post coitum in one female. The number of females affected increased and until day 17 post coitum it was recorded in 18 females in this group.
In group 3, one female had dyspnea on day 8 post coitum followed by breathing noises observed in this female for several days. Breathing noises were also recorded for 7 more females in this group; in 4 animals for one day and in 3 animals for four days.
A red secretion from the nose and eyes was noted in several females in all groups including control. This finding was considered to be related to the treatment route.

GROUP B
All females survived until the scheduled necropsy.
Treatment with the test material caused breathing noises in females in groups 3 and 4.
In group 4, breathing noises were observed on day 3 of the treatment in one female. The number of females affected increased and on day 12 of the treatment it was recorded in all 6 females. The breathing noises were observed until the end of the treatment period and on day 1 of the recovery period. No breathing noises were observed in any female during the remaining recovery period days 2 to 57.
In group 3, breathing noises were observed for the first time on day 5 of treatment. Four females were affected in this group, with breathing noises also observed on day 1 of the recovery period, but not thereafter.
A red secretion from the nose and eyes was noted in several females in all groups including the control. This finding was observed during the treatment and on day 1 of the recovery period but not thereafter. It was considered to be related to the treatment route.

BODY WEIGHTS
GROUP A
Mean body weight gain from day 6 to 21 post coitum was 38.2, 37.2, 32.4 and 29.4 % whereas mean corrected body weight gain was 1.4, 1.9, -2.4 and -5.4 % in groups 1, 2, 3 and 4, respectively.
Treatment with the test material caused a dose dependent body weight loss followed by a reduced body weight gain and a reduction in body weights in groups 3 and 4. A body weight loss of 2 and 5 % was noted in groups 3 and 4, respectively, on day 8 post coitum followed by a reduced body weight gain during the remaining study period. The reduction in body weights as well as the reduction in body weight gain was statistically significant from day 7 to 21 post coitum in both groups. Also corrected body weight gain (body weight gain corrected for the gravid uterus weight at termination) was dose dependently reduced in both groups. After subtraction of the gravid uterus weights, a body weight loss was established with a statistical significance in both groups if expressed as absolute values and in group 4 if expressed as a percentage of the body weight at the start of the treatment.
In group 2, body weight was lower if compared to the control values with a statistical significance during most of the study period, as well as before the start of treatment. Body weight gain in this group was however similar to the control values and corrected body weight gain was slightly higher than the control value. For these reasons lower body weights in group 2 were considered not to be related to the treatment with the test material but due to biological variability.

GROUP B
Treatment with the test material caused a reversible reduction in body weights and body weight gain in group 4. Body weight loss of 8.1 % was noted on day 8 and a reduced body weight gain on day 15 of the treatment period. The reduction in body weight gain was statistically significant during the entire treatment period. After the completion of the treatment, body weight gain recovered and was higher than in the control group with a statistical significance during the entire recovery period. As a consequence, body weights were reduced during the treatment period with a statistical significance on day 8 of this period. Although the body weight gain recovered and increased during the treatment, body weights had not recovered to the pre-dose values by the end of the treatment (day 15). Body weights recovered after the completion of the treatment and were higher than the control values during the recovery period with a statistical significance on day 29.
In groups 2 and 3, no statistically significant differences in body weight gain or body weights were noted if compared to the control group during the treatment. During recovery, a slight but statistically significant increase in body weight gain but with no significant changes in body weights was observed on individual days in both groups. These differences were considered to be incidental.
Mean body weight gain in groups 1, 2, 3 and 4 was, respectively: 3.0, -0.6, 0.2 and -2.6 % during the treatment and 17.9, 24.0, 24.3 and 29.4 % during the recovery period.

FOOD CONSUMPTION
GROUP A
Mean food consumption from day 6 to 21 post coitum was 20.4, 18.9, 17.8 and 16.1 g/animal/ day in groups 1, 2, 3 and 4, respectively.
Treatment with the test material caused a dose dependent reduction in food consumption in groups 3 and 4. The reduction was statistically significant from day 6 to 18 post coitum in both groups. Afterwards it remained lower when compared to the control value; however the differences were not statistically significant.
In group 2, lower food consumption was recorded during the entire study with a statistical significance on days 0 - 3, 6 - 12 and 15 - 18. As the differences in food consumption were already recorded before the start of treatment and remained similar during the study, they were considered not to be related to the treatment with the test material but due to biological variability.

REPRODUCTION DATA
GROUP A
Four females in the control group, three females in group 2 and two females in group 4 were not pregnant. One female in group 2 had implantations only. All remaining females were pregnant and had foetuses at termination on day 21 post coitum.
The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by treatment with the test material. Mean incidence of post-implantation loss per dam was 0.5, 0.9, 0.6 and 0.7, whereas mean number of foetuses per dam at termination was 13.4, 12.1 12.3 and 13.3 in order of ascending dose levels.

GROUP B
Treatment with the test material caused a reversible reduction in food consumption in group 4. The reduction was statistically significant from day 1 to 8 of the treatment period. Afterwards food consumption recovered and was similar to the control value from day 8 to 15 of the treatment. During the recovery period, food consumption was higher than the control values during the first four weeks with a statistical significance from day 8 to 29 of this period and similar to the control values during the remaining four weeks of this period.
In groups 2 and 3, food consumption was not affected by the treatment with the test material. Mean food consumption was 15.4, 14.6, 14.6 and 13.4 g/animal/day during the treatment and 16.7, 17.6, 17.5 and 19.0 g/animal/day during the recovery period in groups 1, 2, 3 and 4, respectively.


MACROSCOPIC PATHOLOGY
GROUP A
In group 4, foci on the lungs were found in two females (nos. 67 and 68). This finding was considered to be test material-related. No further findings were noted during the necropsy in any group.

GROUP B
No findings were observed during macroscopic examination at any dose level.

HISTOPATHOLOGY
GROUP A
Histopathology examination was performed on the lungs from six selected pregnant females per group and from 2 females with macroscopically identified findings in the lungs. Treatment with the test material caused lesions with a dose dependent frequency and severity in groups 3 and 4. Phagocytic alveolar macrophage foci were noted in all six females from group 3 at minimal or slight severity and in all six females from group 4 at slight or moderate severity. Further, granulolymphocytic alveolar inflammation was recorded at minimal degree in four females from group 3 and at minimal to moderate degree in all six females from group 4. The granulolymphocytic alveolar inflammation at minimal degree was recorded also for one female in the control group.
The macroscopically identified foci in two females in group 4 were correlated to alveolar haemorrhage or phagocytic alveolar macrophage foci.
No test material related lesions were found in the lungs of females in group 2.

GROUP B
No test material-related findings were noted during the histopathological examination of female lungs after four or eight weeks of the recovery period. All findings were considered to be within the spontaneous background occurrence of the finding in untreated rats.

Dose descriptor:

NOEL

Effect level:

5 mg/m³ air

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

5 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOEL

Effect level:

15 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

25 mg/m³ air (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
EXTERNAL ABNORMALITIES AND VARIATIONS
No test material-related findings were observed during external examination of the foetuses in any group.
The only finding recorded during external examination of foetuses at termination was haematoma found in five foetuses from litter no. 47 and one foetus from litter no. 48 in group 3. Due to the isolated occurrence and lack of dose dependency, this finding was considered to be incidental.

SEX RATIOS
No effects on the sex ratio of the foetuses were noted in any group.
The proportion of male foetuses was 49.2, 54.6, 47.4 and 49.4 % in order of ascending dose levels.

BODY WEIGHTS
Mean foetal body weights calculated on a litter basis were: 4.8, 4.9, 5.0 and 4.5 g whereas calculated on an individual basis, they were 4.7, 4.8, 4.8 and 4.4 g, both cited in order of ascending dose levels
Treatment with the test material caused a reduction in foetal body weights in group 4. This reduction was statistically significant if calculated on an individual basis and not statistically significant if calculated on a litter basis.
Foetal body weight in groups 2 and 3 were not affected by the treatment with the test material.

VISCERAL ABNORMALITIES AND VARIATIONS
During visceral examination of the foetuses, findings were noted in: 54 % OF examined foetuses (in 100 % of litters) in group 1; 60 % of examined foetuses (in 100 % of litters) in group 2; 49 % of examined foetuses (in 91 % of litters) in group 3; and 58 % of examined foetuses (in 95 % of litters) in group 4.
Treatment with the test material caused an increase in the incidence of large or slightly large foetal thyroid in group 4. This finding was recorded in 12 % of foetuses (in 65 % of litters); 4 % of foetuses (from 20 % of litters) had large thyroid and 8 % of foetuses (from 50 % of litters) had slightly large thyroid. In the control group slightly large thyroid was found in 2 % (in 11 % litters).
The incidence of the large thyroid in group 4 was approximately twice as high as in the historical control group with the highest incidence where 5 % of foetuses (in 29 % of litters) were found with this finding; 2 % of the foetuses (from 10 % of litters) had large thyroid and 3 % of the foetuses (from 24 % of litters) had slightly large thyroid.
The frequency of the remaining findings was within the normal biological background.

MICROSCOPIC EXAMINATION OF THYROIDS
During histopathological examination of the foetal thyroids diffuse follicular hypertrophy and/or hyperplasia at minimal to moderate degree was noted in all five male foetuses from group 4 and at slight or moderate degree in four females from group 4. A minimal degree of this finding was recorded in one group 1 female foetus. Mitotic figures in follicular epithelial cells were increased in both male and female group 4 foetuses.

SKELETAL EXAMINATION
The evaluation of foetuses for skeletal development showed treatment-related changes in group 4 including incomplete or lack of ossification of cervical arch, metatarsals, caudal vertebrae and hind paw phalanges. In addition, the percentage of foetuses with one or more wavy ribs was higher in this group if compared to the control group.
In groups 2 and 3, no findings were recorded which were considered to be test material related.

Dose descriptor:

NOEL

Effect level:

15 mg/m³ air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

visceral malformations

Dose descriptor:

NOAEL

Effect level:

15 mg/m³ air

Based on:

test mat.

Sex:

not specified

Basis for effect level:

visceral malformations

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Summary of Performance of Mated Females

Group	1	2	3	4
Dose (μg/L air)	0	5	15	25
Number of mated females	22	22	22	22
Not pregnant	4	3	0	2
Resorptions only	0	1	0	0
No. females with live foetuses at termination*	18	18	22	20

*Only dams with at least one live foetus at Caesarean section were used for the calculations of food consumption, body weight gain and corrected body weight gain data.

 

Inhalation Technical Data

The achieved group aerosol concentrations were 4.7, 15.1 and 26.0 µg/L.

- Nominal Aerosol Concentration

Group nominal aerosol concentrations are given below (mean ± SD, n = number of exposures, CV = coefficient of variation):

Group 2: 11.2 ± 1.6 µg/L (n = 45, CV = 14.4 %)

Group 3: 34.6 ± 5.1 µg/L (n = 45, CV = 14.7 %)

Group 4: 58.4 ± 11.4 µg/L (n = 45, CV = 19.6 %)

 

-Gravimetric Aerosol Concentrations

The gravimetric aerosol concentrations were stable in groups 3 and 4 during the whole treatment period, based on the small coefficients of variance. There were variations for the aerosol concentrations for group 2 during the treatment period. These fluctuations were considered to be mainly related to differences in the adsorption of water due to the hygroscopic properties of the test material as well as to the loss of moisture from the filter due to the use of dried air for aerosol generation on different days and, therefore, not reflecting real differences in the actual aerosol concentrations. The extent of the water adsorption can be seen from the difference to the corrected gravimetric values. The results are presented in the following table (mean ± SD, n = number of exposures, CV = coefficient of variation):

 

Table 2: Gravimetric Aerosol Concentrations

Group	Group Gravimetric Aerosol Concentration [μg/L]	Corrected Gravimetric Aerosol Concentration [μg/L]
1	7.8 ± 1.8 (n = 24, CV = 23.5 %)	4.7 ± 1.1
2	25.2 ± 3.0 (n = 24, CV = 12.0 %)	15.1 ± 1.8
3	43.4 ± 2.9 (n = 24, CV = 6.7 %)	26.0 ± 1.7

 

- Particle Size Determination

The values for gravimetrically determined Mass Median Aerodynamic Diameter (MMAD) and Geometric Standard Deviation (GSD) were as stated in the following table. The MMADs were at the lower limit of the target range of 1 to 3 μm, therefore deposition of the particles can be assumed to have occurred mainly in the lower but also in the upper respiratory tract. In addition, the Geometric Standard Deviations (GSD) were within the target range of 1.5 to 3. In conclusion, the particle size distribution obtained was considered to be appropriate for this type of study.

Table 3: Gravimetric determination of particle size distribution

Group	Mean MMAD [μm] (mean GSD)	Range of MMAD [μm]	Range of GSD	Number of Determinations	Mass Percentage of Particles <3.0 μm
2	1.63 (2.40)	1.53 - 1.73	2.24 - 2.59	3	75.7
3	2.04 (2.36)	1.86 - 2.14	2.20 - 2.52	3	67.3
4	1.58 (2.26)	1.46 - 1.67	2.23 - 2.26	3	78.4

Conclusions:

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) for the toxicity in pregnant females were considered to be 5 µg/L air. In non-pregnant females, the NOEL for systemic toxicity was established at 15 µg/L air, whereas the NOAEL was established at 25 µg/L air.
Although foetal thyroids were increased in size at 25 µg/L air, a dose which caused adverse maternal toxicity, the causal correlation for these observations was unclear. Also foetal findings at 25 µg/L for the postnatal live young could not be conclusively established as non-treatment related. Therefore the NOEL as well as NOAEL for prenatal developmental toxicity was considered to be 15 µg/L air.

Executive summary:

The potential of the test material to cause prenatal developmental toxicity via the inhalation route was investigated in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700, and Japanese Guideline 12 Nohsan No. 8147 (2-1-18) under GLP conditions.

The purpose of this study was to detect effects on the pregnant Han Wistar rat, development of the embryo and foetus consequent to exposure of the pregnant female via inhalation route (by nose-only, flow-past exposure). A recovery group of non-mated females in all dose groups and the control group were observed for reversibility, persistence or delayed occurrence of systemic toxic effects in the lung.

Four groups of 22 mated females (main study animals) and 6 non mated females (recovery animals) were treated with the test material once daily, for 6 hours per day. Mated females were treated from day 6 post coitum (implantation) to day 20 post coitum (the day prior to Caesarean section) and recovery animals from day 1 to 15 of a concurrent treatment period at target dose levels of 0, 5, 15 and 25 µg/L air (Groups 1, 2, 3 and 4, respectively).

All mated females were sacrificed on day 21 post coitum and the foetuses were removed by Caesarean section. For the recovery animals, three females per group were sacrificed after four weeks and three females per group were sacrificed after eight weeks of the recovery period.

The achieved group aerosol concentrations were 4.7, 15.1 and 26.0 µg/L. The mean mass median aerodynamic diameter (MMAD) was between 1.46 and 2.14 μm for all groups. Therefore, the aerosol was considered to be respirable to rats.

- Main study animals

All females survived until the scheduled necropsy. Treatment with the test material caused breathing noises in eight females in group 3 and eighteen females in group 4. Dyspnea was observed in one female in group 3. No further test material- related findings were noted in any group.

Treatment with the test material caused a dose dependent reduction in body weights, body weight loss followed by a reduced body weight gain and a reduction in corrected body weight gain in groups 3 and 4. These effects were considered to be adverse. No test material-related effects on bodyweights or body weight gain were noted in group 2.

Treatment caused a dose dependent reduction in food consumption in groups 3 and 4. This reduction was statistically significant during the most of the study and was accompanied by reduced body weights, reduced body weight gain during the study and reduced corrected body weight gain at termination at both dose levels and therefore the effect was considered to be adverse. No test material-related effects on food consumption were noted in group 2.

The relevant reproduction data (post-implantation loss and number of foetuses per dam) were not affected by the treatment with the test material.

Treatment with the test material caused foci on the lungs in two females in group 4. Histopathology examination performed on the lungs from six selected pregnant females per group revealed lesions in this organ with a dose dependent frequency and severity in groups 3 and 4: phagocytic alveolar macrophage foci and granulolymphocytic alveolar inflammation. The macroscopically identified foci in two females in group 4 were correlated to alveolar haemorrhage or phagocytic alveolar macrophage foci. No macroscopic or microscopic findings were recorded in group 2.

- Foetal Data

No test material-related findings were noted during the external examination of foetuses and no effects on the sex ratio of the foetuses were noted in any group.

Treatment with the test material caused a reduction in foetal body weights in group 4. This effect was considered not to be adverse. No effects on foetal body weights were noted in groups 2 and 3.

Treatment caused an increase in the incidence of large or slightly large foetal thyroid in group 4. This effect was observed in the presence of maternal toxicity. However, the relationship between the maternal effects and the increased thyroid size remained unclear. The frequency of the remaining findings was within the normal biological background.

Histopathological examination of foetal thyroids revealed that the increased size of the organ in group 4 was correlated with a diffuse follicular hypertrophy/hyperplasia and an increase in mitotic figures in follicular epithelial cells.

Treatment with the test material caused an increased frequency of incomplete ossified or lack of ossification of several bones and an increase in the number of foetuses with wavy ribs. These effects were considered to unlikely have any adverse impact on the post-natal growth and development.

In groups 2 and 3, no findings were recorded which were considered to be test material-related.

- Recovery Females

All females survived until the scheduled necropsy. Treatment with the test material caused breathing noises in females in groups 3 and 4. This finding was observed until day 1 of the recovery period but not thereafter. No further test material- related findings were noted in any group.

Treatment with the test material caused a reversible reduction in body weights and body weight gain in group 4. These effects were considered not to be adverse. Body weights and body weight gain in groups 2 and 3 were considered not to be affected by treatment.

Treatment with the test material at the high-dose level caused a reduction in food consumption with recovery being observed during the treatment. This effect was considered not to be adverse. In groups 2 and 3, food consumption was not affected by the treatment with the test material.

No findings were noted during macroscopic examination of females after four or eight weeks of the recovery period. No test material-related findings were noted during the histopathological examination of female lungs after four or after eight weeks of the recovery period.

Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) for the toxicity in pregnant females were considered to be 5 µg/L air. In non-pregnant females, the NOEL for systemic toxicity was established at 15 µg/L air, whereas the NOAEL was established at 25 µg/L air.

Although foetal thyroids were increased in size at 25 µg/L air, a dose which caused adverse maternal toxicity, the causal correlation for these observations was unclear. Also foetal findings at 25 µg/L for the postnatal live young could not be conclusively established as non-treatment related. Therefore the NOEL as well as NOAEL for prenatal developmental toxicity was considered to be 15 µg/L air.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted on read-across material

Remarks:

Study performed to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results. Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

Justification for type of information:

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

Reason / purpose for cross-reference:

other: Read-across target

Qualifier:

no guideline followed

Principles of method if other than guideline:

Swiss mice were administered subcutaneously with manganese chloride tetrahydrate at doses of 0, 2, 4, 8 and 16 mg/kg/day from gestation day (gd) 6 through to 15. Females were sacrificed on gestation day 18, and foetuses were examined for external, visceral, and skeletal abnormalities.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

Swiss

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Interfauna Ibercia (Barcelona, Spain)
- Weight at study initiation: 28.32 g
- Diet: Panlab rodent chow, ad libitum
- Water: ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 10 %
- Photoperiod (hrs dark / hrs light): 12:12 hour light/dark cycle

Route of administration:

subcutaneous

Vehicle:

physiological saline

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: MnCl2 solutions were prepared fresh daily in 0.9 % saline

VEHICLE
- Amount of vehicle : 0.10 mL

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:2
- Length of cohabitation: Overnight
- Proof of pregnancy: vaginal plug

Duration of treatment / exposure:

9 days

Frequency of treatment:

Daily

Duration of test:

18 days

No. of animals per sex per dose:

20 animals per group

Control animals:

yes, concurrent vehicle

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule: Daily

FOOD CONSUMPTION: Yes
- Time schedule: Monitored daily


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 18
- Organs examined: Liver and kidney and gravid uterus were examined

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No data
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: All live foetuses
- Soft tissue examinations: Yes: One third per litter
- Skeletal examinations: Yes: Two thirds per litter
- Head examinations: No data
- Other: The body weights of all live foetuses were measured

Statistics:

The unit of comparison was the pregnant female of the litter. Kruskal-Wallis analysis of variance procedures were employed to assess the overall effects of MnCl2. Pairwise comparisons were made by the Mann-Whitney U-test. Statistically significant differences between control and test groups were analysed by a two tailed Student's t-test. Significance levels were chosen at P < 0.05.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Treatment related mortality was observed in the high-dose group, 6 of the 19 dams died prior to scheduled necropsy. Statistically significant reductions in body weight and food consumption were noted in the 8 mg/kg group (gd 15-18) and at 16 mg/kg (gd 6-15). A significant decrease in corrected body weight at 8 and 16 mg/kg relative to the controls was noted. Gravid uterine weights were found to be significantly decreased in the 8 and 16 mg/kg/day groups, although the corrected body weight change appeared unaffected by Mn treatment. Relative maternal liver weights were found to be significantly decreased compared to controls at 16 mg/kg.

Dose descriptor:

other: LD50

Effect level:

320 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: other:

Dose descriptor:

NOAEL

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No significant treatment related effects on the number of total implants, early resorptions, dead foetuses or sex ratio were noted. There was a significant increase in the number of late resorptions in the 4, 8 and 16 mg/kg/day groups. There were seven litters with 100 % resorptions in the highest dosing group. A dose response relationship was noted between decreased foetal body weight and increasing dose concentrations. These were found to be significantly below control values in the 8 and 16 mg/kg/day dose groups.

There were no significant increase in the number of litters with one or more affected foetuses in any treatment group in comparison to controls for total external and visceral abnormalities. The incidence of wavy ribs and delayed or reduced ossifications in the sternebra, parietal and occipital were statistically significant (P < 0.05).

Dose descriptor:

NOAEL

Effect level:

2 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Body and organ weights at termination for mice treated with manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. of dams	19	17	17	18	13
Body weight (g)	61.2±4.1	60.7± 8.0	59.3± 8.8	47.2± 6.0^f	44.8± 4.4^f
Gravid uterine weight (g)^a	20.7± 1.8	19.6± 5.3	18.3± 7.8	9.9± 6.0^f	3.4± 1.4^f
Corrected body weight (g)^a	40.5± 1.8	41.1± 3.9	41.0± 2.7	37.3± 3.6^d	41.4± 3.6
Corrected body weigh change (g)^b	8.5± 2.2	7.0± 2.1	6.8± 2.2	6.7± 2.8	8.5± 2.5
Liver weight (g)	3.1± 0.4	3.2± 0.6	3.0± 0.4	2.7± 0.3^e	2.9± 0.2
Relative liver weight (%)^c	7.7± 1.3	7.8± 1.0	7.3± 0.9	7.2± 1.0	7.0± 0.3^d
Kidney weight (g)	0.48± 0.05	0.48± 0.04	0.5± 0.05	0.45± 0.04	0.53± 0.07^d
Relative kidney weight (%)^c	1.18± 0.12	1.17± 0.08	1.21± 0.07	1.21± 0.05	1.28± 0.13^d
Values indicate mean±SD ^aCorrected body weight = bodyweight at termination – gravid uterine weight ^bCorrected body weight change = corrected body weight – body weight on gestational day 0 ^cCalculated as percentage of corrected body weight ^d, e, fSignificantly different from controls (P < 0.05; P < 0.01; P < 0.001, respectively)

Table 2: Gestational parameters and foetal weights in mice foetuses following maternal exposure to manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. of dams	19	17	17	18	13
No. of total implants/litter	13.6 ± 2.5	13.1± 3.0	12.5± 5.1	11.7± 4.6	14.0± 1.6
No. of live foetuses/litter	12.7± 3.3	10.6± 3.0	10.0± 4.7	4.2± 4.0^b	0.3± 0.6^b
No. of non-viable implants/litter
Early resorptions	0.7± 0.7	1.2± 1.0	0.6± 0.6	1.6± 2.0	1.9± 3.1
Late resorptions	0.1± 0.3	0.7± 1.1	1.4± 0.8^a	4.1± 2.8^b	11.6± 4.1^b
Dead foetuses	0.0± 0.0	0.6± 0.9	0.5± 0.7	1.7± 2.7	0.2± 0.3
Sex ratio (m/f)	1.17± 0.96	1.24± 0.62	1.07± 0.72	1.01± 0.50	1.13± 0.81
Average foetal body weight/litter (g)	1.18± 0.13	1.17± 0.09	1.13± 0.12	0.97± 0.11^b	0.82± 0.10^b
Values indicate mean ± SD ^a, bSignificantly different from controls (P < 0.01; P < 0.001 respectively)

 

Table 3: Morphological defects in mice foetuses following maternal exposure to manganese chloride tetrahydrate

	Dose (mg/kg/day)
	0	2	4	8	16
No. foetuses examined viscerally (No. litters)	103 (19)	70 (17)	70 (17)	34 (18)	0 (0)
Enlarged heart
Foetuses affected	0	0	0	4	-
Litters affected	0	0	0	4	-
Renal hypoplasia
Foetuses affected	0	0	2	6^b	-
Litters affected	0	0	2	6	-
No. foetuses examined skeletally (No. litters)	139 (19)	111 (17)	119 (17)	54 (18)	7 (6)
Asymmetrical sternebrae
Foetuses affected	16	19	11	13	0
Litters affected	9	9	6	9	0
Wavy ribs
Foetuses affected	0	2	0	5^c	0
Litters affected	0	2	0	4	0
Dorsal hyperkiphosis
Foetuses affected	0	4	1	1	0
Litters affected	0	1	1	2	0
Sternebrae, delayed ossification
Foetuses affected	0	0	25^c	36^c	7^c
Litters affected	0	0	11^b	18^c	6^c
Parietal bone, reduced ossification
Foetuses affected	0	0	0	6^a	6^c
Litters affected	0	0	0	5	6^c
Occipital bone, reduced ossification
Foetuses affected	0	0	0	6^a	6^c
Litters affected	0	0	0	5	6^c
a, b, c Significantly different from controls (P < 0.05; P < 0.001, respectively)

Conclusions:

Under the conditions of the study the no observable adverse effect level (NOAEL) for maternal toxicity in mice was 4 mg MnCl2.4H2O/kg/day. The NOAEL for embryotoxicity was 2 mg/kg/day, there was no evidence of major malformations at any dosage level used.

Executive summary:

Groups of female mice were administered with 2, 4, 8, or 16 mg/kg of manganese chloride daily from day 6-15 of gestation. Maternal toxicity was observed at the highest dose level with significant mortality (32 %). There was also a significant effect on body weight (decrease), organ weight (liver and kidney), as well as reduced food consumption. The only gestational parameter affected by manganese chloride treatment was an increase in late resorptions. In pups, increasing concentrations of manganese chloride caused decreasing body weight. Relatively minor skeletal effects were also observed.

The conclusions of this study are strengthened by the use of multiple dose groups. Since severe maternal toxicity was observed it cannot be excluded that the effect on late resorptions and on pups is due to this effect.

Since the study was conducted with manganese chloride, which represents a more available form of manganese, rather than with the registered substance itself, the study was assigned a reliability score of 2. Use of data on manganese dichloride is considered to be suitable and more precautionary since manganese dichloride is highly soluble; findings from the study are therefore considered to represent a worst case scenario for inorganic Mn compounds.

 

Reference 3

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Species:

rabbit

Abnormalities:

not specified

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Quality of whole database:

Although there are a number of studies investigated neurochemistry in foetuses/pups pre/postnatally, none are considered to be rounded oral route developmental toxicity studies.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

5 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

The chosen study was conducted to the contemporary OECD 414 guideline and was to GLP.

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subacute

Species:

mouse

Quality of whole database:

The study chosen to represent the dermal route (a subcutaneous study), although not identifiably to GLP, was well described and appeared to follow OECD 414 guideline.

Additional information

The potential of the test material to cause prenatal developmental toxicity via the inhalation route was investigated in rats in accordance with the standardised guidelines OECD 414, EU Method B.31, US EPA OPPTS 870.3700, and Japanese Guideline 12 Nohsan No. 8147 (2-1-18) under GLP conditions. Treatment caused an increase in the incidence of large or slightly large foetal thyroid and diffuse thyroid follicular hypertrophy and/or hyperplasiain at the top dose. This effect has a significant background incidence in control animals, and the maximum severity seen in top dose animals did not exceed the maximimum seen in control. It was also seen the presence of maternal toxicity. In the 2 generation reproduction study (Jardine, 2015), no effect was apparent in pups sacrificed at weaning. Overall it is considered to be a transient effect and of no toxicological relevance. Under the conditions of this study, the NOAEL (No Observed Adverse Effect Level) as well as the NOEL (No Observed Effect Level) for the toxicity in pregnant females were considered to be 5 mg/m3 air based on reduced food consumption and body weight gain, and a reduction in corrected body weight gain in pregnant females. In non-pregnant females, the NOEL for systemic toxicity was established at 15 mg/m3 air, whereas the NOAEL was established at 25 µg/L air, based on the same effects. The NOEL for prenatal developmental toxicity was considered to be 15 µg/L air based on bone ossification effects and wavy ribs which are reversible after birth.

In a developmental toxicity study broadly compliant with OECD 414, female mice were administered either 2, 4, 8, or 16 mg/kg of manganese chloride subcutaneously daily from day 6-15 of gestation. Maternal toxicity was observed at the highest dose level with significant mortality (32%). There was also a significant effect on body weight (decrease), organ weight (liver and kidney), as well as reduced food consumption. The only gestational parameter affected by manganese chloride treatment was an increase in late resorptions. In pups, increasing concentrations of manganese chloride caused decreasing body weight. Relatively minor skeletal effects were also observed. Since severe maternal toxicity was observed it cannot be excluded that the effect on late resorptions and on pups is due to this effect. The NOAEL for for embrytoxicity (late resorptions) was 2 mg/kg bw/day (equivalent to a 200 mg/kg bw/day external dose given dermal absorption of 1%). The NOAEL for maternal effects (reduced body weight gain) was 4 mg/kg bw/day (equivalent to 400 mg/kg bw/day external dose). The NOEL for developmental toxicity (wavy ribs and delayed or reduced ossifications in the sternebra, parietal and occipital bones) was 4 mg/kg bw/day (equivalent to 400 mg/kg bw/day external dose).

Justification for selection of Effect on developmental toxicity: via inhalation route:
A purpose-conducted OECD 414 and GLP compliant developmental toxicity study via the inhalational route.

Justification for selection of Effect on developmental toxicity: via dermal route:
This is a subcutaneous developmental toxicity study, which was carried out to a study plan equivalent to OECD GL 414.

Toxicity to reproduction: other studies

Additional information

The effects of inhlation of MnCl2 on the potential functional and morphological development of the nervous system of rat offspring arising from exposure in utero and during early life were examined in a GLP compliant study conducted in accordance with the standardised guidelines OECD 426 and EPA OPPTS 870.6300.

During the study manganese dichloride was administered by nose only inhalation at actual aerosol concentrations of 3.5, 12.3 and 17.6 μg/L air. Due to a calculation error in the data obtained during the technical trials, the factor was too low and consequently the achieved aerosol concentrations were below target. Measurement of manganese dichloride in the milk showed clearly that the pups were exposed to the test material via milk during lactation.

At the dose level of 17.6 μg/L air, up to 70% of the dams showed breathing noises during the gestation period (starting on day 8 post coitum) with steadily decreasing numbers of affected dams towards the end of the period. During the lactation period no breathing problems occurred, except for three females towards the end of this period. One dam was observed to show signs of severely laboured breathing together with a weakened condition, body weight loss, ruffled fur and a hunched posture; this dam was subsequently killed in extremis on day 15 post coitum.

Dams at dosages of 12.3 and 17.6 μg/L air had reduced mean food consumption directly after treatment commencement. At the mid dose level this effect lasted for one week; within this week mean body weight gain was slightly reduced. Both parameters recovered afterwards and no effects on absolute body weight were observed. At the high dose level, reduction of food consumption lasted for two weeks and was accompanied by a body weight loss of 4% during the first week after treatment. At start of the lactation period, food consumption, body weight and body weight gain recovered at this dose level. The transient effects at 12.3 μg/L air were considered to be not adverse, whereas the effects at 17.6 μg/L air, despite recovery, were considered to be adverse due to the severe clinical signs at one animal. No test material-related effects were noted at 3.5 μg/L air.

Treatment of the dams did not affect the viability or survival of the offspring. However, pups from the high dose group showed slightly reduced mean food consumption from day 21 post partum onwards. Mean body weight gain was dose-dependently reduced in all dose groups after weaning, although this was not always statistically significantly different in the low and mid dose group. Since the reduction in mean body weight gain did not have an effect on absolute mean body weights at the low and mid dose group, the reduction in mean body weight gain was considered to be not adverse in contrast to the high dose group, in which a reduction of 5% was measured.

The retardation in body weight development was not reflected at any other parameters such as developmental indices, sexual maturation or morphometric measurements of the brain.

The daily exposure of the test material, in utero and in early life, produced no behavioural abnormalities or neuropathological findings. All of the histopathological findings encountered were considered to have arisen spontaneously or post mortem.

Based on these findings the NOEL (No Observed Effect Level) for dams and pups was established at 12.3 mg/m3 air.

Justification for classification or non-classification

There is no reliable animal evidence to link MnCl2 with specific and direct reproductive or developmental toxic effect via any relevant routes of exposure. Embryotoxic and developmental effects were considered a result on maternal toxicity including thyroid and ossification effects, which were also completely reversible after birth. No teratogenicity was observed.

No classifcation for reproductive toxicity is proposed.