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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See the read-across report attached in Section 13.
Cross-reference
Reason / purpose:
other: read-across target
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted on read-across material
Justification for type of information:
See the read-across report attached in Section 13.
Reason / purpose:
read-across source
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Critical effects observed:
no
Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Critical effects observed:
no
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Critical effects observed:
no
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed
Critical effects observed:
no
Reproductive effects observed:
no

Data source

Reference
Reference Type:
publication
Title:
A two-generation inhalation reproductive toxicity study upon the exposure to manganese chloride
Author:
McGough D & Jardine L
Year:
2016
Bibliographic source:
Neurotoxicology
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: pink powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
(Crl:CD1(SD))
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: F0 animals commenced treatment at 6–8 weeks of age; F1 animals commenced treatment shortly after weaning for 11 weeks.
- Housing: Rats were housed in cages. Cages were racked by treatment group with males and females racked separately.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: The F0 animals were allowed to acclimate for 13 days before the commencement of dosing.
- For at least 7 days prior to the commencement of dosing, all animals were conditioned to the restraint procedures (to adapt to the inhalation chamber) used for nose-only exposure by placing the animals in the restraint tubes for gradually increasing periods of restraint time up to the maximum expected duration to be used on the study—6 h daily.
- Each batch of diet was routinely analysed by the supplier for various nutritional components and chemical and microbiological contaminants, including manganese, which is a known essential element in the SDS Rat and Mouse diet and the levels of manganese were (84–94 mg/kg) in each batch of diet. The drinking water was periodically analysed for dissolved materials, heavy metals, pesticide residues, pH, nitrates, nitrites and selected bacteria. Results of these analyses did not provide evidence of contamination; however, Mn is a known component of potable water and was present at a concentration of 1.9 mg Mn/L. Therefore additional Mn exposure from diet and water intake is expected.

Administration / exposure

Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
nose only
Vehicle:
air
Details on exposure:
GENERATION OF TEST ATMOSPHERE
- System of generating particulates/aerosols: The test material was a pure pink powder, it was passed through a centrifugal grinder using the finest mesh available and then sieved using a mesh the size of 100 or 180 mm prior to use. Test material aerosols were generated using a Wright Dust Feed generator device.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: up to 14 days
- 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.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Target Concentration (mg/L Air) - Nose only: 0, 5, 10 and 20 µg/L air
F0 Actual Concentration (mg/L Air) - Nose only: 0, 6, 15 and 25 µg/L air
F1 Actual Concentration (mg/L Air) - Nose only: 0, 4, 10 and 17 µg/L air

The overall aerosol concentrations aerosols were 6, 15 and 25 µg/L for the F0 Generation and 4, 10 and 17 µg/L for the F1 Generation. This was considered to be satisfactorily close to target concentrations of 5, 10 and 20 µg/L for Groups 2, 3 and 4, respectively. For the air-alone control group (Group 1), the test material was nondetectable or non-quantifiable for all samples collected over the course of the study.
The particle size distribution (mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD)) investigations assured that the test aerosols were respirable to the animals and that good pulmonary exposure was achieved. Particle size distribution investigations assured that the test aerosols were respirable to the animals and that good pulmonary exposure was achieved.
Duration of treatment / exposure:
6 hours per day/ 7 days a week
Frequency of treatment:
F0 animals were treated for 10 weeks prior to pairing for mating. Daily exposure was for 6 h, 7 days a week. Meanwhile F1 animals commenced treatment shortly after weaning for 11 weeks (prior to mating). Daily exposure was for 6 h, 7 days a week. For F0 and F1 males, this treatment continued until the day prior to termination (a total of 17 weeks per generation).
For F0 and F1 females, the animals were dosed throughout gestation up to and including Day 19 of gestation. The animals were not dosed from Day 20/21 of gestation 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: for 1 h per day
From Day 3–4 of lactation: for 2 h per day
From Days 5–20 of lactation until prior to termination (ca Day 21 of lactation): for 6 h per day.
Animals that did not produce litters, continued on a 6 h dosing until the scheduled termination. Animals that had a litter loss continued on a 6 h dosing regimen until scheduled sacrifice.
Details on study schedule:
- From each treatment group, at least 24 males and 24 females were retained for post weaning assessments. These animals continued on the study (F1) and were dosed for approximately 11 weeks (6 h daily) after weaning, and throughout mating, gestation and lactation until termination after the F2 generation had reached Day 21 of lactation.
Doses / concentrationsopen allclose all
Dose / conc.:
5 other: µg/L air
Dose / conc.:
10 other: µg/L air
Dose / conc.:
20 other: µg/L air
No. of animals per sex per dose:
F0: 28 animals per sex per dose
F1: at least 24 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected after a nose-only inhalation exposure dose range finder study. Ten females and 10 males per group were exposure to the test material at 5, 20 and 30 µg/L. The study was scheduled for 9 weeks however; exposure was stopped at 3 weeks for the high dose as adverse clinical signs included crackling/gasping respiration resulted in the premature sacrifice of 3 animals. Necropsy findings included distended intestines, froth filled trachea, discoloured lungs. Exposure continued to the end of the study for the low and mid dose animals with minimal effects. The high dose for the main study was therefore reduced to 20 µg/L.
- Rationale for animal assignment: In the main study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: YES

OTHER:
Blood samples were taken from the tail vein of all adult animals for analysis of Mn concentrations prior to dosing, prior to mating and prior to weaning/necropsy. Samples were collected from all F0 generation animals at pre-dose, prior to mating and prior to weaning/necropsy. Meanwhile for the F1 generation, samples were collected from all animals (timing and volume depended on weight of animals, prior to mating and at necropsy. All samples were stored at -80 °C and analysed for Mn in whole blood via ICP-MS.
Oestrous cyclicity (parental animals):
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.
Sperm parameters (parental animals):
A detailed qualitative examination of the testes was made, taking into account the tubular stages of the spermatogenic cycle. Computer assisted sperm analysis was also conducted to assess the motility of the sperm. Sperm counts using a haemocytometer to obtain a total sperm count was assessed. A sperm smear was prepared and stained with eosin and evaluated for morphological abnormalities. One testis was decapsulated and homogenized and the homogenisation resistant spermatids were counted using a haemocytometer.
Litter observations:
- 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.
- 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 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
- ca Day 21 of lactation
- Animals that did not produce litters, continued on a 6 h dosing until the scheduled termination. Animals that had a litter loss continued on a 6 h dosing regimen until scheduled sacrifice.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The tissues assigned to histological examination were processed; sections were cut 4, stained with haematoxylin and eosin (H&E) and evaluated by light microscopy from animals in the Controls and High dose groups. Additionally, a Periodic Acid Schiff and Haematoxylin (PAS-H) stained section was prepared from the left testis.
- The following organs were collected and preserved in 10 % neutral buffered formalin, unless otherwise indicated: Animal identification, Lymph node, bronchial, Brainb Lymph node, cervical, Epididymis x 2, Nasal cavity, Gland, adrenal x 2,Ovary x 2, Gland, pituitary, Pharynx, Gland, prostate, Spleen, Gland, seminal vesicle, Testes (Davidson's fixative), Gland, thyroid x 2, Trachea (anterior), Kidney x 2, Trachea, (posterior), Larynx, Uterus, Liver, Vagina and Lung.
Postmortem examinations (offspring):
SACRIFICE
- Where practicable, any pups that were found dead or were killed during lactation were sexed and examined. 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.

HISTOPATHOLOGY / ORGAN WEIGTHS
- 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 Haematoxylin and Eosin (H&E) section was cut, stained and evaluated
Statistics:
- Where required to assist with interpretation, tests were applied to determine the statistical significance of observed differences between Control and groups receiving the test material. Unless otherwise stated, all statistical tests were two-sided and performed at the 5 % significance level using in house (validated) 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 stabilise 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, and by analysis of covariance (ANCOVA) using terminal body weight as the covariate.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
One female animal in Group 3 had clinical signs including wheezing, unkempt coat, walking on tip toes, rolling gait and weight loss recorded over Days 83–90 of the study. Due to the signs dosing for the animal was stopped for a two days. However, the animal recovered from these signs and dosing continued until scheduled termination. As no similar findings were noted in the other animals in this group or in group 4, these signs were considered to be incidental. Blood concentrations correlated with exposure levels in males and females) confirming incremental systemic test material availability.
At target 20 µg/L (group 4) there were 2/28 males noted as having wheezing respiration.
Other clinical signs noted in the F0 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Four animals in the F0 generation (one Control, 2 at target 5 µg/L and one at 10 µg/L) were killed prematurely for various reasons –difficulties giving birth, reduced activity, partially closed eyes, unkempt coat, open lesion on tail etc.
There was no treatment-related pattern to these deaths and these were not attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At target dose 20 µg/L, there was a decrease in body weight gain in males over Days 0–21 of the study (29 % lower gains than those of the Controls). 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 dose 20 µg/L, the 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 (9 % of the Controls). Gains over the lactation period were similar to the controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At target dose 20 µg/L, there was reduced food consumption for males throughout the majority of the study, compared with the controls.
At target dose 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 dose 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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Pre treatment manganese levels in all F0 animals were similar to controls.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related findings observed in the reproductive tract in the F0 generation. Histological findings related to treatment were confined to the respiratory tract as follows:

- Larynx: In the larynx there was a broadly dose-related minimal to moderate squamous metaplasia with minimal to moderate submucosal inflammation. Minimal to marked ulceration of the laryngeal epithelium was associated with the
squamous metaplasia in several animals from all treated groups. Occasional incidences of mineralisation, intraluminal necrotic debris or intra-epithelial pustules were seen in some of the treated animals.

- F0 lungs: In the lungs, the principal test material-related change was seen in centroacinar regions where there was minimal or mild inflammation and focal or diffuse minimal or mild bronchoalveolar hyperplasia. This latter finding was considered reactive. Minimal or mild goblet cell hyperplasia in the bronchial or bronchiolar epithelium was present in animals exposed to target 20 µg/L together with occasional incidences of degeneration and/ or squamous metaplasia of the bronchiolar epithelium. Minimal inflammatory findings (inflammatory cell foci and perivascular inflammatory cell infiltration) were also present with a greater incidence in animals exposed to the test material than in controls.

- Nasal cavity: In the nasal cavity, minimal or mild goblet cell hyperplasia and minimal to moderate eosinophilic globules in the olfactory epithelium were observed in all the male treated groups and in females exposed to target 10 or 20 µg/L. At all dose levels, there was a greater incidence of minimal or mild submucosal inflammatory cell infiltration compared to controls. In males, inflammation of the nasolacrimal duct and squamous metaplasia of the ductal epithelium was seen in most animals exposed to target 10 or 20 µg/L. In addition to these changes, incidences of minimal or mild focal degeneration of the olfactory, respiratory or transitional epithelia, minimal or mild atrophy of the olfactory epithelium, ulceration and focal squamous metaplasia were observed, mainly in animals exposed to target 10 or 20 µg/L, but occasionally in animals at target 5 µg/L. Deposits of crystalline material, presumed to be the test material, was seen in the nasolacrimal ducts of a few animals in the treated groups.

- Pharynx: Minimal goblet cell hyperplasia was observed in the pharynx of most males exposed to target 20 µg/L and there were occasional incidences of minimal or mild focal epithelial degeneration, focal inflammation and focal squamous metaplasia in males exposed to the test material at 10 or 20 µg/L.

- Trachea: In the trachea, minimal or mild focal squamous metaplasia and inflammation at the carina and minimal or mild focal epithelial degeneration at sites other than the carina were observed at all dose levels.

- Other microscopic findings observed were considered incidental, or of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to the inhalation of the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The stages of the oestrus cycles and their mean duration were similar in all Test groups compared to the control group for both generations.
There was no treatment-related effect on the follicle type or incidence in either generation. The scoring methodology included the quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on the sperm motility, count of morphology at any of the dose levels applied, or in either generation.
Reproductive performance:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on mating performance, fertility or duration of gestation in either F0 or F1 generation.

Litter size and pup mortality
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 historical background range, hence these increases were considered to be incidental.

Litter and pup weights
- In all treated groups, group mean litter and pup weights were comparable to the controls.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical observations noted in the F1 animals were considered to be incidental or due to the dosing procedure (wet, unkempt coat).
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
5 animals in the F1 generation were (one Control, 3 at target 10 µg/L and 1 at target 20 µg/L) killed prematurely for various reasons –difficulties giving birth, reduced activity, partially closed eyes, unkempt coat, open lesion on tail etc. There was no treatment-related pattern to these deaths and these
were not attributed to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At target dose 20 µg/L, there was a reduction in group mean body weight gain of the males during the first 5 days of the study (p < 0.001), 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 lactating females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At target dose 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 (ranges from p < 0.05 to p < 0.001).
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
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.
The F1 generation pre-treatment Mn blood levels were higher than the control with dose dependent correlation indicating exposure prior to treatment via lactation. Milk Mn levels were not measured directly but Mn blood pre-treatment levels confirm Mn exposure prior to weaning.
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Kidneys: At target doses 5 and 10 µg/L, kidney weights in males were statistically higher than the control (p < 0.01 and p < 0.05, respectively), however there was no dose-response relationship to this increase and following covariance analysis with body weight, these findings were no longer evident.
- At target doses 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 with body weight.

Weanling 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 with body weight, this reduction did not achieve statistical significance. There were no effects on any organ weights at target doses of 5 and 10 µg/L.
Gross pathological findings:
no effects observed
Description (incidence and severity):
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 of the test material.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
There were no treatment related findings observed in the reproductive tract in the F1 generation.
Histological findings related to treatment were confined to the respiratory tract as follows:

- Nasal cavity: Crystals were occasionally observed in the nasal cavity and in the pharynx from animals exposed to target 10 or 20 µg/L. They consisted of small amounts of needle-shaped crystals either deposited on the olfactory epithelium in the nasal cavity, or free in the lumen of the pharynx. These crystals were considered to result from deposition of the test material in some parts of the respiratory tract. Squamous metaplasia in the nasal cavity was observed mainly in the nasolacrimal duct and to a lesser extent in the transitional and respiratory epithelia.

- Trachea: In the trachea, findings such as epithelial degeneration, squamous metaplasia and submucosal inflammation were observed predominantly in the carina.

- Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat, and/or were of similar incidence and severity in control and treated animals and, therefore, were considered unrelated to administration of the test material.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The stages of the oestrus cycles and their mean duration were similar in all Test groups compared to the control group for both generations.
There was no treatment-related effect on the follicle type or incidence in either generation. The scoring methodology included the quantification of the primordial and growing oocytes, and the confirmation of the presence or absence of the corpora lutea.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- There were no treatment-related effects on the sperm motility, count of morphology at any of the dose levels applied, or in either generation.
Reproductive performance:
no effects observed
Description (incidence and severity):
- There were no treatment-related effects on mating performance, fertility or duration of gestation in either F0 or F1 generation.

Effect levels (P1)

Dose descriptor:
NOAEL
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects observed

Target system / organ toxicity (P1)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
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 historical background range, hence these increases were considered to be incidental.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
In all treated groups, group mean litter and pup weights were comparable to the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
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.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
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.
Gross pathological findings:
no effects observed
Description (incidence and severity):
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 of the test material.
Histopathological findings:
no effects observed
Description (incidence and severity):
There were no treatment related findings observed in the tissues examined of the F1 or F2 weanlings.
Other effects:
no effects observed
Description (incidence and severity):
The type and distribution of observations amongst pups did not indicate any association with treatment if compared to species historic data.

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed

Target system / organ toxicity (F1)

Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Description (incidence and severity):
At target 20 µg/L, group mean litter weights were slightly lower than the controls; this was considered a reflection of the smaller litter size and not an adverse effect. This interpretation was supported by the mean pup weights in both males and females being comparable to the controls.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Weanling organ weights
- F1 generation, F2 production: Slight intergroup differences in organ weights did not achieve statistical significance and were not attributed to treatment.
Gross pathological findings:
no effects observed
Description (incidence and severity):
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 of the test material.
Histopathological findings:
not examined
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
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.
- The type and distribution of observations amongst pups did not indicate any association with treatment if compared to species historic data.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 other: µg/L air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related effects were observed

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level of 20 µg/L.
Based on these findings, the test material could not be considered a reprotoxicant under these conditions of exposure. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation cannot be considered as reprotoxicants.
Executive summary:

The reproductive toxicity of the test material was investigated in accordance with the standardised guidelines OECD 416 and OPPTS 870.3800.

Doses were selected after a nose-only inhalation exposure dose range finder study. Ten females and 10 males per group were exposure to manganese chloride at 5, 20 and 30 µg/L. The study was scheduled for 9 weeks however; exposure was stopped at 3 weeks for the high dose as adverse clinical signs included crackling/gasping respiration resulted in the premature sacrifice of 3 animals. Necropsy findings included distended intestines, froth filled trachea, discoloured lungs. Exposure continued to the end of the study for the low and mid dose animals with minimal effects. The high dose for the main study was therefore reduced to 20 µg/L.

In the main study, F0 animals were randomised into 3 test groups and one control group, each containing 28 males and 28 females. These animals were dosed for 10 weeks (6 h daily) 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 the study (F1) and were dosed for approximately 11 weeks (6 h daily) after weaning, and 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 sperms.

There were no deaths related to treatment, though respiratory tract effects were observed in F0 animals in the mid and high dose animals. Body weight and food consumption were affected at high dose in both generation. There were no treatment-related effects on the oestrous cycles, mating performance, sexual maturity, fertility or duration of gestation or litter size, the sperm motility, count of morphology (sperm) or the ovary follicle scoring in either generation.

Under the conditions of this study, the No Observed Effect Level (NOEL) for reproductive performance was considered to be the target dose level of 20 µg/L.

Based on these findings, the test material could not be considered a reprotoxicant under these conditions of exposure. Therefore, soluble and insoluble forms of inorganic manganese compounds by extrapolation cannot be considered as reprotoxicants.