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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-Jul to 02-Sep-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well performed GLP and OECD Guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Manganese Oxalate, dihydrate
IUPAC Name:
Manganese Oxalate, dihydrate

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
TEST ANIMALS

- Animals: Rat, RccHanTM: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Number of Animals: 44 males (11 per group) and 44 females (11 per group)
- Age (at Start of Treatment): 11 weeks
- Body Weight Range (at Start of Treatment): 309 to 341 g (males) and 188 to 225 g (females)
- Identification: Cage card and individual animal number (ear tattoo) for parental animals. On day 1 post partum, pups were individually tattooed with Indian ink.
- Randomization: Computer-generated random algorithm. In addition body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
- Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS

- Conditions: Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 22 ± 3 °C; relative humidity range: 30 - 70%). The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.
- Accommodation: Individually in Makrolon type-3 cages with wire mesh tops and sterilized standard softwood bedding (‘Lignocel’ J.Rettenmaier & Söhne GmbH & CoKG, 73494 Rosenberg / Germany, imported by Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) with paper enrichment (ISO-BLOX from Harlan Laboratories B.V., Netherlands), batch/lot nos. 02105120301, 77-100099. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.
- Diet: Pelleted standard Harlan Teklad 2914C (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland) was available ad libitum (batch no 20/12). The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. A bacteriological assay, chemical and contaminant analyses of respective samples were performed.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Details on exposure:
DOSE FORMULATIONS

The dose formulations were prepared weekly.

Based upon the results of dose formulation analyses performed during a non-GLP dose range finding study (Harlan Laboratories Study), the stability of the test item formulations was considered to be sufficient to justify weekly preparation.

MANGANESE OXALATE, DIHYDRATE was weighed into a glass beaker on a tared precision balance and the vehicle was added (w/v). Using a magnetic stirrer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, any remaining vehicle was added if necessary.

Separate formulations were prepared for each concentration.

Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATION

Dose formulations were stored at room temperature (20 ± 5 °C) in glass beakers, protected from light.

Based upon the results of stability analyses performed within the non-GLP Harlan Laboratories study (Implementation and validation of an analytical method for dose formulation analysis), dose formulations were stable for at least four weeks when stored in these conditions.

TREATMENT

- Method: Oral, by gavage
- Rationale for Method: Administration by gavage is a common and accepted route of exposure for this type of studies.
- Frequency of Administration: Once daily
- Daily Target Dose Level: 0 mg/kg/day (Group 1), 100 mg/kg/day (Group 2), 300 mg/kg/day (Group 3) and 1000 mg/kg/day (Group 4)
- Rationale for Dose Level Selection: The dose levels were selected based on a previous dose range-finding toxicity study in Han Wistar rats (Harlan Laboratories Study) .
- Dose Volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD

The test item was used as the analytical standard. Representative samples were dispatched to the analytical laboratories internally (under ambient condition) and either stored frozen at -20 ± 5 °C until analysis or directly analyzed.

RESULTS

The linearity of the analytical systems used for sample analyses was demonstrated with a good relationship between peak areas measured and working standard concentrations. All calibration points used met the acceptance limit of ±20% variation from the calibration curve derived by linear regression analysis. The regression coefficients (R2) calculated were found to be better than 0.99.

The MANGANESE OXALATE, DIHYDRATE peak was assigned in sample chromatograms by comparison to that of working standards. In blank sample chromatograms no significant peak appeared at the retention time of MANGANESE OXALATE, DIHYDRATE and, therefore, the absence of the test item in the vehicle control samples (0.5% aqueous solution of carboxymethylcellulose (CMC)) was confirmed.

The application formulations investigated during the study were found to comprise MANGANESE OXALATE, DIHYDRATE in the range of 91.6% to 118.8% and, thus, the required content limit of ±20% with reference to the nominal content was met. The homogeneous distribution of MANGANESE OXALATE, DIHYDRATE in the preparations was approved because single results found did not deviate more than 2.8% (acceptance criterion: <15%) from the corresponding mean.

In addition, the test item was found to be stable in application formulations when kept up to eight days at room temperature due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean, except for group 2 that exceeded the acceptance criteria. However, as no degradation was observed, the formulation is considered to be stable, although not fulfilled the acceptance criterion (±10%)).

In conclusion, the results indicate the accurate use of the test item MANGANESE OXALATE, DIHYDRATE and 0.5% CMC as vehicle during this study. Application formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.
Details on mating procedure:
During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. 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.

If a female did not mate during the 14-day pairing period, this female was paired with a male of the same group which had already mated successfully. If mating was not recorded during this additional pairing period of a maximum of 14 days, the female was sacrificed and, if indicated, the reproductive organs examined histopathologically in order to ascertain the reason for the infertility.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 was designated as the day on which a female had delivered all her pups.
Duration of treatment / exposure:
Minimum 4 weeks (males) and approximately 7 weeks (females)
Frequency of treatment:
Once daily
Duration of test:
MALES

- Acclimatization: 8 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days
- Treatment Ends: On day before sacrifice
- Necropsy: After treatment for at least 28 days, when no longer needed for assessment of reproductive effects

FEMALES

- Acclimatization: 8 days
- First Test Item Administration: Day 1 of pre-pairing
- Pre-Pairing: 14 days
- Pairing: 14 days maximum
- Gestation: Approximately 21 days
- Treatment Ends: On day 4 post partum
- Necropsy: On day 5 post partum (pups on day 4 post partum)
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
nominal conc.
in terms of Manganese Oxalate, anhydrous form (= submission substance)
No. of animals per sex per dose:
11
Control animals:
yes, concurrent vehicle
Details on study design:
The purpose of this study was to generate preliminary information on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provided information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

This study should provide information to assess the need to conduct further investigations and may provide guidance in the design of subsequent studies.

Rationale for Choice of Species, Route of Administration and Dose Levels: The rat is a suitable species for repeated dose and reproduction/developmental toxicity studies required by regulatory authorities. The oral route is one possible route for human exposure.

Dose levels were selected in agreement with the Sponsor, based on the results of a non-GLP dose range-finding study (Harlan Laboratories study).

Examinations

Maternal examinations:
VIABILITY/MORTALITY

Twice daily

CLINICAL SIGNS

Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION

Pre-Pairing period days 1 - 8 and 8 - 14; gestation days 0 - 7, 7 - 14 and 14 - 21 post coitum, and lactation days 1 - 4.
No food consumption was recorded during the pairing period.

BODY WEIGHTS

Recorded daily from treatment start to day of necropsy.

TERMINATION AND NECROPSY

Dams were sacrificed on day 4 post partum.

All animals sacrificed were subjected to a detailed macroscopic examination. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution. At the scheduled sacrifice, all animals were sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred.

For the parent animals, special attention was directed at the organs of the reproductive system. The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites.

TISSUE PRESERVATION

The ovaries from all parental females were preserved in neutral phosphate buffered 4% formaldehyde solution.

HISTOTECHNIQUE

All organ and tissue samples from animals to be examined by the principal investigator for histopathology phase were processed, embedded and cut at an approximate thickness of 2 - 4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the principal investigator.

HISTOPATHOLOGY

Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the principal investigator. The same applied to all occurring gross lesions and to all animals which died spontaneously or had to be terminated in extremis.

A histopathology peer review was performed.
Ovaries and uterine content:
The ovaries and the uterus were examined after termination on day 4 post partum. Examinations included the number of corpora lutea and the number of implantation sites.
Fetal examinations:
Not performed
Statistics:
The following statistical methods were used to analyze food consumption, body weights and reproduction data:

- Means and standard deviations of various data were calculated.
- 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 dichotomized without loss of information.
Indices:
From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation losses and mean litter size, dead/live pups at first litter check, pups sex ratio and viability indices.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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

Details on maternal toxic effects:
1. IN-LIFE DATA

VIABILITY/MORTALITY

One female (no. 77) treated with 300 mg/kg/day was sacrificed for ethical reasons on Day 3 of the pre-pairing period. A second female (no. 74) treated with 300 mg/kg/day was found dead on Day 3 of the pre-pairing period. All females survived the lactation period.

DETAILED CLINICAL OBSERVATIONS (DAILY)

There were no clinical signs of toxicological relevance noted in the test item-treated females.

In the single female treated with 300 mg/kg/day which was found dead on day 3 of pre-pairing, breathing noises were noted on days 2 and 3, followed by ruffled fur, hunched posture and convulsion on day 3. The cause of death was considered to be accidental aspiration of dosing solution and was not considered to be a toxicological effect.

FOOD CONSUMPTION

Pre-Pairing, Gestation and Lactation Periods: The mean daily food consumption of the females was unaffected by treatment during the pre-pairing, gestation and lactation periods.

BODY WEIGHS

Pre-Pairing, Pairing, Gestation and Lactation Periods: During the pre-pairing period, the mean body weights of the test item-treated females compared favorably with those of the control females. During the gestation period, the mean body weights of the test item-treated mated females were similar to those of the control females. During the lactation period, the mean body weights of the test item-treated females that gave birth were unaffected by treatment.

2. TERMINAL FINDINGS

MACROSCOPICAL FINDINGS

In the survivors, there were no gross lesions that could be attributed to treatment with the test item. All findings recorded were considered to be within the range of normal background alterations.

In female no. 77 that was killed in extremis, dark-red discoloration was recorded in the lung. This was deemed to correlate with the hemorrhagic lesions caused by accidental influx of the doing solution containing the test item. No gross lesions were observed in the one female (no. 74) that died prematurely.

HISTOPATHOLOGY FINDINGS

- Findings in Decedents: In female no. 77, alveolar hemorrhage with inflammatory cell infiltrate, intra-alveolar/bronchial flocculent and crystalloid substances and focal necrosis in the bronchiolar-alveolar duct region were observed in the lung. These findings were considered to be due to an accidental influx of the dosing solution and were considered to be the cause of the animal’s morbidity.

- Findings in Surviving Rats: There were no histomorphologic lesions that could be attributed to treatment with the test item. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: in terms of Manganese Oxalate, anhydrous form (= submission substance) , equivalent to 1252 mg/kg bw. of the test item (Manganese Oxalate, dihydrate)
Basis for effect level:
other: other:
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: in terms of Manganese Oxalate, anhydrous form (= submission substance) , equivalent to 1252 mg/kg bw. of the test item (Manganese Oxalate, dihydrate)
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Examination of pups revealed no embryotoxic or teratogenic effects

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
other: in terms of Manganese Oxalate, anhydrous form (= submission substance) , equivalent to 1252 mg/kg bw. of the test item (Manganese Oxalate, dihydrate)
Remarks on result:
other: no advers effect observed

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

1. REPRODUCTION, BREEDING AND PUP DATA

 

SUMMARY OF PERFORMANCE

 

P Animals Breeding for F1 Litters

 

Group
(mg/kg/day)

1
(0)

2
(100)

3
(300)

4
(1000)

Female numbers

45 - 55

56 - 66

67 - 77

78 - 88

Number of females paired

11

11

9

11

Number of females mated

11

11

9

11

Number of females not pregnant

0

0

0

0

Number of females which reared their pups until day 4 post partum

11

11

9

11

 

MATING PERFORMANCE AND FERTILITY

 

No effect on mating performance or fertility was observed at any dose level.

 

DURATION OF GESTATION

 

The duration of gestation was similar at all dose levels.

 

CORPORA LUTEA COUNT

 

The number of corpora lutea observed at all dose levels compared favorably with those of the controls.

 

IMPLANTATION RATE AND POST-IMPLANTATION LOSS

 

The implantation rate of the test item-treated and control groups compared favorably.

 

Although the mean post-implantation loss was marginally higher in females treated with 1000 mg/kg/day when compared with the control females, the females treated with 100 mg/kg/day and 300 mg/kg/day were lower than the control females. The mean number of affected litters were similar

 

LITTER SIZE AT FIRST LITTER CHECK

 

The mean litter sizes were similar in test item-treated and control females.

  

POST NATAL LOSS DAYS 0 - 4 POST PARTUM

 

At 300 and 1000 mg/kg/day, females showed higher post-natal losses (as a percent of living pups) and greater numbers of affected litters than the control females, there was no clear dose-response relationship. Significantly greater numbers of affected litters (p<0.05) and the total number of affected pups (p<0.01) were noted in litters at 300 mg/kg/day when compared with the controls. At 1000 mg/kg/day, these values were higher than those of the controls but did not attain statistical significance and were therefore considered to be of no toxicological relevance.

 

The average number of living pups on day 4 post partum were similar in all groups.

 

EXTERNAL EXAMINATION OF PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

One pup (no 1) of litter 73 (300 mg/kg/day) and two pups (nos 6 and 9) of litter 81 (1000 mg/kg/day) were found dead at first litter check.

 

These mortality rates are within the normal range of variation and are therefore not considered to be treatment related.

 

At first litter check, no abnormal findings were noted in surviving pups of control dams or in dams treated with 100 mg/kg/day. At 300 mg/kg/day, one pup was found without visible milk in the stomach. This finding was also recorded for two pups (one each in two litters) at 1000 mg/kg/day.

 

On day 4 post partum, there were no abnormal findings noted in surviving pups at any dose level.

 

PUP SEX RATIOS

 

The sex ratios at first litter check were within the range of typical variation.

 

BODY WEIGHTS OF PUPS TO DAY 4 POST PARTUM

 

The mean body weights of the pups on day 4 post partum were considered to be unaffected by the treatment with the test item.

 

MACROSCOPICAL FINDINGS OF PUPS

 

There were no macroscopical findings in any pup necropsied on day 4 post partum at any dose level, nor were any macroscopical findings seen in pups found dead at first litter check.

At 300 and 1000 mg/kg/day, females showed higher post-natal losses (as a percent of living pups) and greater numbers of affected litters than the control females, there was no clear dose-response relationship. Significantly greater numbers of affected litters (p<0.05) and the total number of affected pups (p<0.01) were noted in litters at 300 mg/kg/day when compared with the controls. At 1000 mg/kg/day, these values were higher than those of the controls but did not attain statistical significance and were therefore considered to be of no toxicological relevance.

 

The average number of living pups on day 4 post partum were similar in all groups.

 

2. IN-LIFE DATA OF PARENTAL MALES

 

VIABILITY/MORTALITY

 

One male (no. 30) treated with 300 mg/kg/day was found dead on day 2 of pre-pairing. All remaining parent males survived until their respective scheduled day of necropsy.

 

DETAILED CLINICAL OBSERVATIONS (DAILY)

 

During days 4-6 of the pre-pairing period, control male no. 7 showed visible weight loss, ruffled fur or breathing noises. During days 2-8 of pre-pairing, male no. 15 treated with 100 mg/kg/day showed ruffled fur and breathing noises. At 300 mg/kg/day, ruffled fur was recorded on day 8 of the pre-pairing period in males 24, 25, 29 and 33. This finding was also noted on day 8 in males 35, 38, 39, 41, 42 and 43 treated with 1000 mg/kg/day, and also in the latter male on day 9. These findings were not considered to be of toxicological relevance.

 

There were no clinical signs noted in the test item-treated males during the pairing period.

 

FOOD CONSUMPTION

 

Pre-Pairing Period: The mean daily food consumption of the test item treated males was similar to that of the control males.

 

BODY WEIGHTS

 

Pre-Pairing and Pairing Periods: During the pre-pairing and pairing periods, the mean body weights of the test item-treated males were generally similar to those of the control males. The mean body weight gain values were also unaffected.

 

3. TERMINAL FINDINGS OF PARENTAL MALES

 

ORGAN WEIGHTS

 

In males, the mean absolute and relative organs weights were unaffected by the treatment with the test item.

 

MACROSCOPICAL FINDINGS

 

In the survivors, there were no gross lesions that could be attributed to treatment with the test item. All findings recorded were considered to be within the range of normal background alterations.

 

No gross lesions were observed in the male (no. 30) that died prematurely.

 

HISTOPATHOLOGY FINDINGS

 

- Findings in Decedents: In male animal no. 30, no relevant changes were noted in the testes, epididymides, prostate or seminal vesicles, although in a state of progressive autolysis. In female animal no. 74, postmortem congestion was observed in ovaries. The histomorphologic evidence identifying the exact cause of death were not obtained in these animals because of the limited number of organs/tissues examined. Nonetheless these animals’ deaths were considered not to be the effects of the treatment with the test item, because the death and morbidity did not happen in animals treated with 1000 mg/kg/day.

 

- Findings in Surviving Rats: There were no histomorphologic lesions that could be attributed to treatment with the test item. All microscopic findings recorded were within the range of normal background lesions which may be recorded in animals of this strain and age.

 

- Testes - Detailed Examination Including Sperm Staging: The stages were checked on completeness of cell populations, completeness of stages and degenerative changes. No differences on the completeness of stages or cell populations of the testes were recorded between the control animals and the high dose animals. As a result of the detailed examination of the testis, it was judged that there were no test item-related effects on the testicular histomorphology, including spermatogenesis and interstitial cell structure.

Applicant's summary and conclusion

Conclusions:
This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage administration of the test item MANGANESE OXALATE, DIHYDRATE to rats. MANGANESE OXALATE, DIHYDRATE was administered in 0.5% aqueous solution of carboxymethylcellulose as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day (in terms of Manganese Oxalate, anhydrous form, which is equivalent to 125.2, 375.6 and 1252 mg/kg body weight/day of the test item), and controls received the vehicle only. MANGANESE OXALATE, DIHYDRATE was administered to male rats in total for at least 28 days (14 days prior to pairing) and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

One male and one female treated with 300 mg/kg/day were found dead. A second female treated with 300 mg/kg/day was sacrificed for ethical reasons. All remaining animals survived until their respective scheduled day of necropsy.

No test item-related clinical signs or findings were noted in males or females at any dose level. Food consumption, body weights and body weight gain were not affected in males or females up to the dose level of 1000 mg/kg/day.

No effects on mating performance, fertility, corpora lutea count, duration of gestation, implantation rate and post-implantation loss as well as litter size and post-natal loss were noted at any dose level. No test item-related findings in pups or effects on pup body weights were noted at any dose level.

There were no test item-related macroscopical or microscopical findings of toxicological relevance at any dose level.

The NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg/day in terms of Manganese Oxalate, anhydrous form.
Executive summary:

The purpose of this study was to generate preliminary information concerning the effects of MANGANESE OXALATE, DIHYDRATE on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

 

MANGANESE OXALATE, DIHYDRATE was administered to male rats for at least 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

 

The following dose levels in terms of Manganese Oxalate, anhydrous form were applied:

 

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

Control animals were dosed with the vehicle (0.5% aqueous solution of carboxymethylcellulose) alone.

 

The following results were obtained:

 

MORTALITY AND GENERAL TOLERABILITY OF PARENTAL ANIMALS

 

One male and one female treated with 300 mg/kg/day were found dead on days 2 and 3, respectively, of the pre-pairing period. One female treated with 300 mg/kg/day was sacrificed for ethical reasons on Day 3 of the pre-pairing period.

 

FOOD CONSUMPTION OF PARENTAL ANIMALS

 

There were no effects upon the mean daily food consumption of males or females.

 

BODY WEIGHTS OF PARENTAL ANIMALS

 

There were no effects upon the mean body weights of males or females.

 

REPRODUCTION AND BREEDING DATA

 

No effect on mating performance or fertility was observed at any dose level. The duration of gestation was similar at all dose levels.

 

The number of corpora lutea and implantation rate were unaffected in all groups. Minor differences in the mean post-implantation loss were considered to be unrelated to the treatment with the test item.

 

The mean litter sizes were similar in test item-treated and control females. Although females at 300 and 1000 mg/kg/day showed higher post-natal losses, there was no clear dose-response relationship, and were therefore considered to be of no toxicological relevance. The average number of living pups on day 4 post partum were similar in all groups.

 

ORGAN WEIGHTS OF PARENTAL ANIMALS

 

In males, the mean absolute and relative organs weights were unaffected by the treatment with the test item.

 

MACROSCOPICAL FINDINGS AND HISTOPATHOLOGICAL EXAMINATION OF PARENTAL ANIMALS

 

Males: No gross lesions of toxicological relevance were observed in the single male that died prematurely or in any surviving male.

 

Females: In the one female that was killed in extremis, hemorrhagic lesions in the lung were likely caused by accidental influx of the dosing solution containing the test item. No gross lesions were observed in the one female that died prematurely. All remaining females were without macroscopical findings.

 

FINDINGS IN PUPS AT FIRST LITTER CHECK AND DURING LACTATION

 

At 300 mg/kg/day, one pup was found dead at first litter check. At 1000 mg/kg/day, two pups (in one litter) were found dead at first litter check.

 

These mortality rates are within the normal range of variation and are therefore not considered to be treatment related.

 

At 300 mg/kg/day, one pup was found without visible milk in the stomach during the first litter check. This finding was also recorded for two pups (one each in two litters) at 1000 mg/kg/day.

 

On day 4 post partum, there were no abnormal findings noted in surviving pups at any dose level.

 

PUP WEIGHTS TO DAY 4 POST PARTUM

 

The mean body weights of the pups on day 4 post partum were considered to be unaffected by the treatment with the test item.

 

MACROSCOPICAL FINDINGS OF PUPS

 

There were no macroscopical findings in any pup necropsied on day 4 post partum at any dose level, nor were any macroscopical findings seen in pups found dead at first litter check.

 

CONCLUSION

 

The NOEL (No Observed Effect Level) and the NOAEL (No Observed Adverse Effect Level) for general toxicity in males and females and for reproduction/developmental toxicity was considered to be 1000 mg/kg/dayin terms of Manganese Oxalate, anhydrous form.

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