Reaction mass of Magnesium peroxide and magnesium carbonate and magnesium hydroxide and magnesium oxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

other: combined 28-day rep. dose reproduction

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

Read CSR in Section 13 for RA justification

Reason / purpose for cross-reference:

read-across source

Control animals:

yes, concurrent no treatment

Clinical signs:

no effects observed

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:

no effects observed

Other effects:

not specified

Reproductive performance:

no effects observed

Concentrations values were recalculated based on the MW of one of constituents of the reaction mass, MgO2(target substance).

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period. No clinical signs of toxicity were noted during the observation period. Incidental findings that were noted in single females included alopecia or piloerection. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

BODY WEIGHTS: Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

REPRODUCTIVE PERFORMANCE: No toxicologically relevant effects on gestation index and duration, parturation, maternal care and early postnatal pup development ( mortality, clinical signs, body weight and macroscopy) were observed.
The gestation index was 100% for all groups and the duration of gestation was similar between control and treated groups.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

ORGAN WEIGHTS: No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. THe significantly lower absolute thymus weight observed for females at 960.73 mg/kg was considered to be due to a low weight for female no.72. Individual weights of other females of this dose group remained within the range observed among other treated females.

GROSS PATHOLOGY: Animals survivng to the scheduled necropsy were deeply anaesthetised using iso-flurane vapour and subsquently exsanguinated. All animals were subjected to a macroscopic examination with special attention being paid to the reproductive organs. Description of all macroscopic abnormalities were recorded.

HISTOPATHOLOGY : The following slides were examined by a pathologist:
-The preserved organs and tissues of the selected 5 animals/sex groups 1 and 4
-The additional slides of the testes of the selected 5 males of groups 1 and 4 to examine staging of spermatogenesis
-All gross lesions of all animals.

FUNCTIONAL OBSERVATIONS: Hearing ability,pupillary reflex, static righting reflex and grip strength were normal in all animals. The variation in motor activity did not indicate a relation with treatment.

FOOD CONSUMPTION: Food consumption before or after allowance for body weight was similiar between treated and control animals.

HAEMOTOLOGY: No toxicologically relevant changes occurred in haematological parameters of treated rats. Any statistically significant changes were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

MACROSCOPIC EXAMINATION: Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

CLINICAL BIOCHEMISTRY: The following statistically significant changes in clinical biochemistry parameters distinguished treated animals from control animals:
-lower total protein levels in males at 317.04 and 960.73 mg/kg,
-lower albumin levels in males at 960.73 mg/kg,
-lower calcium levels in males at 317.04 and 960.73 mg/kg.
Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain.

URINALYSIS:
The following statistically significant changes in urinary parameters distinguished treated males from control males:
-Lower sodiumexcretion (mmol/TPV) at 317.04 and 960.73 mg/kg,
-Lower potassium excretion (mmol/TPV) at 960.73 mg/kg,
-Higher calcium concentration (mmol/L) at 960.73 mg/kg.

Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. the significant higher specific gravity seen at 317.04 mg/kg was not considered to be toxicologically relevant as it occurred in the absence of a treatment-related trend.

Dose descriptor:

NOAEL

Remarks:

No observed adverse effect level

Effect level:

>= 2 381 mg/kg bw/day

Based on:

other: Recalculated based on the amount of Mg(OH)2 that is present in the reaction mass.

Sex:

male/female

Basis for effect level:

other: No reproduction/developmental toxicity was observed at any dose level

Remarks on result:

other: Generation not specified

Clinical signs:

no effects 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:

no effects observed

Histopathological findings:

no effects observed

Concentrations values were recalculated based on the MW of one of constituents of the reaction mass, MgO2(target substance).

MORTALITY: Two pups of the control group, three pups at 106.68 mg/kg, and one pup at 317,04 mg/kg were found dead or missing during lactation. the missing pups were most likely cannibalised. No pups were found dead or missing at 960,73 mg/kg. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

CLINICAL SIGNS: Incidental clinical symptoms of pups consisted of blue spot on the back and scabbing of the snout or back. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

BODY WEIGHT:Body weights of pups were considered to be unaffected by treatment.

MACROSCOPY: Incidental macroscopic findings for pups that were found dead included autolysis and absence of milk in the stomach. Scabbing on the snout was noted for one surviving pup. The nature and incidence of these findings remained within the range considered normal for pups of this age, and was therefore considered to be of no toxicological relevance.

Dose descriptor:

NOAEC

Generation:

F1

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: all endpoints

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

not specified

Conclusions:

No reproduction/developmental toxicity were observed at any dose level tested with magnesium hydroxide. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined. This dose descriptor is recalculated based on the amount of Mg(OH)2 that is present in the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium peroxide, and yields an NOAEL of 2381 mg/kg bw/d for the reaction mass.

Executive summary:

A number of clinical biochemistry and urinary changes were noted at 317.04 and 960.73 mg/kg in males which included lower total protein, albumin and calcium levels in blood, and lower sodium and potassium excretion and higher calcium concentration in urine. Means of these changes only just exceeded or remained within the range considered normal for rats of this age and strain. Moreover, there were no histopathological correlates in eg. liver or kidneys that would support these changes. Therefore, these changes were considered not to be of toxicological relevance.

Overall, no toxicologically relevant changes were noted in any of the parental parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

Furthermore, no reproduction/developmental toxicity were observed at any dose level. based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.

This dose descriptor is recalculated based on the amount of Mg(OH)2 that is present in the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium peroxide, and yields an NOAEL of 2381 mg/kg bw/d for the reaction mass.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In order to be systemically available, a chemical needs to be absorbed, either via the oral, inhalatory or dermal route. Dissolution of solids is generally assumed to be a prerequisite for absorption. As the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium oxide and magnesium peroxide is a solid inorganic multi-constituent substance, this means that Mg²⁺, OH^-and hydrogen peroxide are the species to be taken into account when assessing its long-term systemic toxicity.

 

* H₂O₂: In the REACH registration dossier for hydrogen peroxide, it has been shown that hydrogen peroxide is rapidly metabolised in the body to oxygen and water and does not bio-accumulate. In none of the repeated dose studies described in the dossier hydrogen peroxide causes directly systemic effects. It is also deemed improbable that hydrogen peroxide would reach inner organs such as ovaries and testes as well as foetuses to cause reproductive toxicity. It is concluded that reproductive toxicity studies will not provide any useful information for the risk assessment of hydrogen peroxide and should therefore not be conducted due to animal welfare reasons.

 

* Mg(OH)₂: For magnesium hydroxide, a combined repeated dose reproductive toxicity screening study (van Otterdijk, 2010) is available. The test is performed according to GLP and OECD guidelines. In this experiment, no effects on male and female fertility and no effects on embryo or fetal development parameters were observed up to the highest tested dose of 1000 mg/kg bw/d. Based on these results, a parental, reproduction and developmental NOAEL of at least 1000 mg/kg bw/d was determined.

 

Based on the above information, it can be concluded that the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium oxide and magnesium peroxide will not exert any reprotoxic effects in vivo.

Justification for selection of Effect on fertility via oral route:
Key study available for read-across substance hydrogen peroxide.

Effects on developmental toxicity

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-03-2010 to 14-05-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conduced according to guidelines.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3550( Reproduction/Developmental Toxicity Screening Test July 200)

Deviations:

no

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

-Source: Charles River laboratories France, L’Arbresle Cedex, France.
-Age at study initiation: Approximately 11 weeks
-Housing:
Lactation: Pups were kept with the dam until termination in Macrolon cages (MIII type, height 18cm)
General: Sterilised sawdust as bedding material and paper as cage enrichment. During activity monitoring animals were housed individually in Macrolon cages with sterilised sawdust as bedding material. No cage-enrichment was provided during activity monitoring.
Diet: Free access to pelleted rodent diet.
Water: Free access to tap water
-Acclimation period: At least 5 days prior to the start of treatment

Environmental Conditions:
Animals were housed in a controlled environment.
Temperature (°C): 21±3°C (actual range 19.7- 21.7°C)
-Humidity (%): A relative humidity of 40-70% (actual range: 34-73%)
- Air changes per hour: 15 air changes per hour
-Photoperiod (Hrs dark/hrs light): 12 hours artificial light and 12 hours darkness per day.

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Accuracy and homogeneity were determined for formulations prepared for use during treatment. Duplicate samples( approx 500 mg), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 50ml. For determination of accuracy, samples were taken at 50% height or at 90%, 50% and 10% height. The latter set of samples was also used for the determination of the homogeneity of the samples. The volumetric flasks were filled up to the mark with 4% aqueous HNO3. The solutions were further diluted with 4% aqueous HNO3 to obtain concentrations within the calibration range.

Details on mating procedure:

- M/F ratio per cage: Following a minimum of 14 days of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating
- Length of cohabitation: Once mating had occurred, the males and females were separated
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged: Females were individually housed in Macrolon cages (MIII type, height 18cm). the females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found complete (i.e. membranes,placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.

Duration of treatment / exposure:

Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-45 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. Females 41, 46, 48, 49 (group 1, table 1), 53, 59 (group 2, table 1), 61, 62, 68 (group3, table 1) and 76 (group 4, table 1) were not dosed during littering.

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.

No. of animals per sex per dose:

Four groups of ten male and ten female Wistar (Hans) rats were exposed by oral gavage to the test substance.

Control animals:

yes, concurrent no treatment

Details on study design:

-Dose selection rationale: Based on the results of a 10-day dose range finding study.
-Rationale for animal selection: This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies.

Maternal examinations:

Detailed clinical Observations;
Time schedule: Daily detailed clinical observations were made in all animals immediately after dosing. Once, prior to the start of treatment and at weekly intervals this was also performed outside the home cage in a standard arena. Arena observations were not performed when the animals were mating, or housed individually.

Functional Observations:
The following tests were performed on the selected 5 animals/sex/group:
-hearing ability, pupillary reflex, static righting reflex and grip strength.
-motor activity test.
During the motor activity test, males were caged individually and females were caged with their pups. The selected males were tested during week 4 of treatment and the selected females were tested during lactation. In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 minutes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes (Table 1)
- Number of implantations: Yes (Table 1)
All animals were subjected to macroscopic examination, with special attention being paid to reproductive organs. Descriptions of all macroscopic abnormalities were recorded. The numbers of former implantation sites and corpora lutea were recorded for all paired females.

Fetal examinations:

-External examinations: Yes:
Each litter was examined to determine the following if possible:
Mortality/ Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Statistics:

For each group the following calculations were performed:
Mating (%) - (Number of females mated/Number of females paired) x 100
Fertility Index (%) – (Number of pregnant females/Number of females paired) x 100
Conception index (%) – (Number of pregnant females/Number of females mated) x 100
Gestation index (%) – (Number of females bearing live pups/ Number of pregnant females) x 100
Duration of gestation – Number of days between confirmation of mating and the beginning of parturition.
Percentage live males at first litter check – (Number of live male pups at first litter check/ Number of live pups at First Litter Check) x 100
Percentage live females at First Litter Check – (Number of live female pups at First Litter Check/ Number of live pups at First Litter Check) x 100
Percentage of postnatal loss Days 0-4 of lactation – (Number of dead pups on Day 4 of lactation/ Number of live pups at First Litter Check) x 100
Viability index (%) – Number of live pups on Day 4 of lactation/ Number of pups born alive) x 100

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Treatment related:

not specified

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macropsy) were observed.

Gestation:

The gestation index was 100% for all groups and the duration of gestation was similar between control and treated groups.

Parturition/ Maternal care: No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.

Early postnatal pup development: The number of dead and living pups at first litter check, postnatal loss, viability index and sex ratio were unaffected by treatment, and clinical signs, body weight and external macroscopy did not reveal treatment-related findings.

Mortality: Two pups of the control group, three pups at 110 mg/kg, and one pup at 330 mg/kg were found dead or missing during lactation. The missing pups were most likely cannibalized. No pups were found dead or missing at 1000 mg/kg. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose related trend and remained within the range considered normal for puppies of this age.

Clinical signs: Incidental clinical symptoms of pups consisted of blue spot on the back and scabbing of the snout or back. The nature and incidence of these clinical signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Body weights: body weights of pups were considered to have been unaffected by treatment.

 

Table 1:

Corpora Lutea and Implantation sites:

Females F0-Generation

 

		Group 1 Control	Group 2 110 mg/kg	Group 3 330 mg/kg	Group 4 1000 mg/kg
Corpora lutea	Mean	14.0	15.1	14.7	14.5
	St.Dev	2.2	2.7	4.4	2.7
	N	10	10	10	10
Implantation sites	Mean	12.6	13.1	12.6	13.2
	St.Dev	1.2	1.6	1.2	1.4
	N	10	10	10	10

Table 2:

Developmental data:

F0- generation- Lactation

 

	Group 1 Control	Group 2 110 mg/kg	Group 3 330 mg/kg	Group 4 1000 mg/kg
Litters
Total	10	10	10	10
Duration of gestation
Mean (+)	21.3	21.3	21.3	21.2
St. Dev	0.5	0.5	0.5	0.4
N	10	10	10	10
Dead pups at first litter check
Litters affected (#)	0	1	1	0
Total	0	1	1	0
Mean	0.0	0.1	0.1	0.0
St.Dev	0.0	0.3	0.3	0.0
N	10	10	10	10
Living pups at first litter check
% of Males/Females	45/55	51/49	55/45	54/46
Total	116	121	118	123
Mean	11.6	12.1	11.8	12.3
St.Dev	1.1	1.4	1.5	1.8
N	10	10	10	10
Postnatal loss
% of Living Pups	1.7	1.7	0.0	0.0
Litters affected	2	2	0	0
Total	2	2	0	0
Mean	0.2	0.2	0.0	0.0
St.Dev	0.4	0.4	0.0	0.0
N	10	10	10	10
Viability Index	98.3	98.3	100.0	100.0

 

Conclusions:

No reproduction/developmental toxicity were observed at any dose level. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg/day was determined.

Executive summary:

Overall, no toxicologically relevant changes were noted in any of the parental parameters investigated in this study.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In order to be systemically available, a chemical needs to be absorbed, either via the oral, inhalatory or dermal route. Dissolution of solids is generally assumed to be a prerequisite for absorption. As the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium oxide and magnesium peroxide is a solid inorganic multi-constituent substance, this means that Mg²⁺, OH^-and hydrogen peroxide are the species to be taken into account when assessing its long-term systemic toxicity.

 

* H₂O₂: In the REACH registration dossier for hydrogen peroxide, it has been shown that hydrogen peroxide is rapidly metabolised in the body to oxygen and water and does not bio-accumulate. In none of the repeated dose studies described in the dossier hydrogen peroxide causes directly systemic effects. It is also deemed improbable that hydrogen peroxide would reach inner organs such as ovaries and testes as well as foetuses to cause reproductive toxicity. It is concluded that reproductive toxicity studies will not provide any useful information for the risk assessment of hydrogen peroxide and should therefore not be conducted due to animal welfare reasons.

 

* Mg(OH)₂: For magnesium hydroxide, a combined repeated dose reproductive toxicity screening study (van Otterdijk, 2010) is available. The test is performed according to GLP and OECD guidelines. In this experiment, no effects on male and female fertility and no effects on embryo or fetal development parameters were observed up to the highest tested dose of 1000 mg/kg bw/d. Based on these results, a parental, reproduction and developmental NOAEL of at least 1000 mg/kg bw/d was determined.

 

Based on the above information, it can be concluded that the reaction mass of magnesium carbonate and magnesium hydroxide and magnesium oxide and magnesium peroxide will not exert any reprotoxic effects in vivo.

Justification for selection of Effect on developmental toxicity: via oral route:
Key study available for read-across substance magnesium hydroxide.

Justification for classification or non-classification

In accordance to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for toxicity to reproduction based on the available information for read-across substances hydrogen peroxide and magnesium hydroxide.