Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Key study: OECD Guideline 415. GLP study. Based on the read-across approach from the analogue MIBKO, the NOAEL for the F1 generation and reproductive toxicity was estimated to be >90.84 mg/kg bw/day (based on no effects on reproduction or developmental parameters or in the F1 pups).

Supporting study: OECD Guideline 407. GLP study. The NOAEL for toxicity on reproductive organs or tissues after a 28 days of oral exposure was determined to be >200 mg/kg bw/day since no effects were observed up to the highest dose tested.

Link to relevant study records

Referenceopen allclose all

Endpoint:
one-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 11, 2007 - April 21, 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Test method according to OECD 415. GLP study.
Qualifier:
according to guideline
Guideline:
OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: (P) 6 wks, (F1) 3 wks.
- Weight at study initiation: (P) Males: 165 to 245 g; Females: 125 to 197 g
- Fasting period before study: Yes
- Housing: Animals were housed in groups of 4 per sex in solid polycarbonate cages with Lignocel 3/4 wood flakes for breeding. During mating 1, male and 1 female from the same treatment group were housed together. During gestation and lactation, females were housed individually or with their litters.
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23 ºC
- Humidity (%): 40-70 %
- Photoperiod (hrs dark / hrs light): Artificial lighting 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The required volume of vehicle was measured and added to the test substance and mixed using a whirlmixer for a minimum of one minute and then stirred magnetically whilst sampling (five minutes). Moisture was avoided at all times during formulation. The test substance was used as supplied. All formulations were prepared freshly each week and stored refrigerated until the day of use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): In a previous study corn oil formulations were shown to be homogenous in the vehicle and stable at ambient temperature for two days and for 15 days following refrigerated storage.
- Concentration in vehicle: 0 (control), 5.0, 15.0, 50.0 mg/mL
- Amount of vehicle (if gavage): 2 mL/kg
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: two weeks
- Proof of pregnancy: Ejected copulation plugs. Vaginal smears were prepared and examined for the presence of spermatozoa and the stage of the oestruous cycle. Evidence of mating was referred to as day 0 of pregnancy
- After 7 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- After successful mating each pregnant female was caged (how): Individually during gestation and with litters during post partum
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration for Weeks 1, 5, 11 and 14 of treatment of the F0 animals and Week 1 of treatment of the selected F1 animals were analysed for achieved concentration of the test substance. The analytical procedure was validated with respect to linearity of detector response, precision of injection, specificity of chromatographic analysis, limit of detection, accuracy and precision. The homogeneity and stability was confirmed for MIBKO in corn oil formulations at a nominal concentration of 50 mg/mL. The mean concentrations of MIBKO in test formulations analysed for the study were within +10%/-15% of nominal concentrations, confirming accurate formulation.
Duration of treatment / exposure:
Parental rats were dosed 10 weeks prior to mating, for up to 2 weeks during mating, or until signs of mating were noted, and then during both gestation and lactation. Selected F1 animals were treated from weaning (Day 21 of age) to approximately 7 weeks of age.
Frequency of treatment:
Daily
Details on study schedule:
- Age at mating of the mated animals in the study: 16 weeks (6 weeks at study initiation + 10 weeks dosing period).
- Offspring were weaned on Day 21 of age and separated from the dam. The allocation of offspring to form the F1 generation was made before weaning on Day 21 of age.
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
No. of animals per sex per dose:
F0 gneration: 28 animals per sex and per dose.
F1 generation: 24 animals per sex and per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study with reference to preliminary work with this compound performed by Huntingdon (NBJ0037/070047). MIBKO at dose levels of 10, 30 and 100 mg/kg/day had no adverse effects on reproductive performance or F1 pre- and postnatal development, following treatment of F0 males and F0 females for 28 days before pairing, and during gestation and post partum.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS:
- Time schedule: at least twice daily
- Observations: evidence of ill-health or reaction to treatment

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Weeks 1 to 2, twice weekly during Weeks 3 to 5 (middle and end of each week) and once a week from Week 6 onwards for F0 females on Days 0, 6, 13 and 20 after mating and Days 1, 7, 14 and 21 post partum. A more detailed physical examination was performed on each adult F0 animal weekly, F0 females on Days 0, 6, 13 and 20 after mating and Days 1, 7, 14 and 21 post partum.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until termination. The weight of each F0 female was recorded weekly until mating was detected. After mating
bodyweights were recorded on Days 0, 3, 6, 10, 14, 17 and 20 and on Days 1, 4, 7, 11, 14, 18 and 21 post partum.

FOOD CONSUMPTION:
- Time schedule: For each F0 male the weight of food supplied, that remaining and an estimate of any spilled was recorded weekly until paired for mating. For each F0 female, the weight of food supplied, that remaining and an estimate of any spilled was recorded weekly until paired for mating, Days 0-2, 3-5, 6-9, 10-13, 14-16 and 17-19 after mating and 1-3, 4-6, 7-10, 11-13, 14-17 and 18-20 post partum.
Oestrous cyclicity (parental animals):
Three weeks prior to mating, vaginal smears were prepared to observe the estrous cycle of the F0 and F1 females. Daily vaginal smears taken from females before necropsy (Days 25 to 28 post partum) were used to determine the stage of the oestrous cycle at termination. For females whose litters had previously died, the smears were taken on the theoretical Days 25-28.
Sperm parameters (parental animals):
Sperm analysis was performed in all male parental generations. After termination, the left vas deferens, epididymis and testis was removed and the epididymis and testis were weighed. The following tests were performed: Sperm motilitly, sperm morphology, sperm count and homogenisation-resistan spermatids count.
Litter observations:
STANDARDISATION OF LITTERS
- On postnatal day 4, litters were culled to 8 or 10 pups, with equal numbers of males and females were possible, by random selection. Pups that were selected continued in the study until postnatal day 55.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
- Following delivery the number of pups were determined and the ratio of male to female pups was calculated.
- Litters were examined daily for dead pups.
- Pups were weight on postanal days 1, 4, 7, 11, 14, 18, 21, 25, and 28.
- Total litter size, number of male and female pups, number of stillborn and live births and grossly malformed pups, and pups showing abnormalities were recorded on postanatal day 1.
- The number of live and dead pups, as well as pups showing malformations or abnormalities, was recorded on postnatal days 4, 7, 14 and 21.
- Sexual maturation: Males were examined daily from day 38 until blanopreputial separation occurred and selected females were examined daily from day 28 until the vaginal opening occurred. Body weights were recorded at these events.

GROSS EXAMINATION OF DEAD PUPS:
- Yes, for external and internal abnormalities.



STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Parturition observation and gestation lengh: umbers of live and dead offspring, duration of gestation.
- Clinical observations: All litters were examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter. Selected F1 animals were assessed weekly from Day 21 of age for detailed physical examinations.
- Bodyweight: Individual offspring bodyweights were recorded on Days 1, 4 (before culling), 7, 11, 14, 18 and 21 of age. The weight of each selected F1 animal was recorded on Days 21, 25 and 28 of age.
- Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
- Sex ratio: The sex ratio of each litter was recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
- Sexual maturation: For all selected males, sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. For all selected females, sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred.

[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, other:]

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities (see below); possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals were killed when majority of litters had been weaned (approx. 16 weeks).
- Maternal animals: Females surviving until the end of the scheduled study period were killed on Day 28 post partum, which failed to produce viable litter on Day 25 and whose litter died before Day 21 post partium on the day the last offspring died.

GROSS NECROPSY
- Gross necropsy consisted of:full macroscopic examination of the tissues, all external features and orífices; the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera, abnormal position, morphology or interaction; external and cut surfaces of the
organs and tissues; abnormalities in the appearance or size of any organ and tissue. For females, the numbers of implantation sites in each uterine horn was counted.

ORGAN WEIGHTS:
- Adrenals, brain, epididymides, kidneys, liver, ovaries, pituitary, prostate, seminal vesicles, spleen, testes, thyroids, uterus with cervix and oviducts.

HISTOPATHOLOGY
- All parental animals from control and 100 mg/kg bw/day.
- Pathology: adrenals, brain, epididymis, kidneys, liver, mammary area-caudal, ovaries, piruitary, prostate, seminal vesicles, spleen, testis, thyroids, uterus with cervix and oviductus, vagina, any abnormal tissue plus the lymph nodes draining the regions adjacent to masses. Reproductive organs were also analysed in animals at 10 and 30 mg/kg bw/day that showed reduced fertility. Spleen (both sexes) and kidneys (males only) were examined for all F0 adults since were considered to exhibit a reaction to treatment at the high dose.
- Histology: Epididymis, kidneys, liver, ovaries, seminal vesicles, uterus, vagina.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as F1 generation were sacrificed approximately at 21 days of age.
- Selected F1 animals were killed at approximately 7 weeks of age.
- Selected F1 offspring + non selected ones with external abnormalities + premature deaths, were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of: full macroscopic examination of the tissues, all external features and orífices; the neck and associated tissues and the thoracic, abdominal and pelvic cavities and their viscera, abnormal position, morphology or interaction; external and cut surfaces of the
organs and tissues; abnormalities in the appearance or size of any organ and tissue. For females, the numbers of implantation sites in each uterine horn was counted.

ORGAN WEIGHTS:
- Unselected F1 offsprings: Brain, spleen and thymus
- Selected F1 offsprings: Spleen

HISTOPATHOLOGY / ORGAN WEIGTHS
- Unselected F1 offsprings: Brain, spleen and thymus.
- Setected F1 offsprings: Spleen.
Statistics:
Statistical analyses were performed by using one or multiple methods, as appropriate. For categorical data, Fisher’s exact probability test was used. For continuous data, one-way analysis of variance (ANOVA), followed by either Dunnett’s multiple comparison or Barlett’s test, was used. Kruskal-Wallis nonparametric ANOVA, followed by the Mann-Whitney U-test, was used for nonparametric data. In addition, postimplantation loss and sex ratio were analyzed by the mixed linear model Wald chi-square test.
Reproductive indices:
Percentage mating, conception rate, fertility index
Offspring viability indices:
Litter size, survival indices (post-implantation survival index, live birth index, viability index, lactation index), sex ratio (percentage males).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Salivation and chin rubbing were the only clinical signs attributed to administration of the test article. These effects were observed in 100 mg/kg bw/day group males and females and diminished following 4 weeks of treatment. No animals were found in moribund conditions. One female at 30 mg/kg/day had a total litter loss with no pups born alive. No remarkable clinical signs were observed. One female was sacrificed for welfare reasons on day 1 of lactation. Clinical signs included piloerection, hunched posture, skin pallor, and hair loss. Neither of these deaths was considered to be treatment related. One female in the 100-mg/kg group was killed on day 1 of lactation due to poor clinical condition.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male at 30 mg/kg bw/day was found dead on day 32, and another male at 100 mg/kg bw/day was found dead on day 112. One control female was found dead on day 35. The cause of dead was not determined.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight, body-weight change was similar to controls at all time points.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was similar to controls at all time points.
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
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):
In the spleens, minimal to moderate hemosiderosis was observed in higher incidence and severity in the 100 mg/kg/day males and females. Minimal congestion was seen in the majority of animals of both sexes treated at 100 mg/kg/day. In the kidneys of males, minimal to moderate cortical tubules with hyaline droplets were seen at a significantly higher incidence in animals treated at 30 mg/kg/day or 100 mg/kg/day. All other findings were considered to be incidental and unrelated to the test substance.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
No effects on estrus cycle were observed.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects on sperm counts or morphology were observed.
Reproductive performance:
no effects observed
Description (incidence and severity):
No effects on precoital time, the number of pregnant females, fecundity, or duration of gestation were seen.
One female each from the control, 10 mg/kg/day, and 100 mg/kg/day groups were not pregnant.
Key result
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
30 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on no effects at the highest dose tested
Critical effects observed:
not specified
Clinical signs:
no effects observed
Description (incidence and severity):
No remarkable clinical signs were observed in litter. A higher incidence of chin rubbing, salivation and post salivation staining was seen in both the males and females at 100 mg/kg/day in F1 generation.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
Litter data, as assessed by the mean number of implantations, total litter size and live litter size on Day 1 and subsequent litter size to Day 21 of age, were similar to the Control and were considered unaffected by treatment. In utero and offspring survival as assessed by post implantation survival index, live birth index, viability index and post partum index was considered unaffected by treatment with MIBKO.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Bodyweight at birth and subsequent weight gain to Day 21 were considered unaffected by treatment.
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):
There was no effect of treatment with MIBKO on the time of completion of vaginal opening or preputial separation at any dose.
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Absolute and bodyweight relative spleen weights were statistically significantly high amongst the offspring of females at 100 mg/kg/day. There were no other obvious effects of treatment for animals receiving 10, 30 or 100 mg/kg/day. Nevertheless, there was no effect of treatment apparent upon the weights of the spleens of F1 generation animals.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No findings were considered related to treatment of the F0 generation.
Histopathological findings:
no effects observed
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Remarks:
(F1 toxicity)
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on no effects at the highest dose tested
Key result
Dose descriptor:
NOAEL
Remarks:
(developmental and sexual maturation)
Generation:
F1
Effect level:
> 100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on no effects at the highest dose tested
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
In a one-generation reproduction study performed with methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats, the NOAEL for parental toxicity was considered to be 30 mg/kg bw/day (based on histopathological effects on the spleen) and the NOAEL for the F1 generation and reproductive toxicity was considered to be >100 mg/kg bw/day (based on no effects observed on reproduction or developmental parameters).
Executive summary:

A one-generation reproduction study was performed according to OECD guideline 415 with the analogue substance methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats. 28 parental rats per sex and per dose, were given methyl isobutyl ketoxime in water by gavage at doses of 0 (control), 10, 30 and 100 mg/kg bw/day during 10 weeks prior to mating, for up to 2 weeks during mating, or until signs of mating were noted. Males were killed following mating, and females were continuously exposed through gestation and lactation. Parental and litter examinations as well as reproduction observations were performed. All surviving male and female parent rats, were sacrificed for postmortem examinations. All stillborn pups, pups found dead, or pups terminated in a moribund condition and those for which the sexual maturation was observed, were subjected to a throrough necropsy. No adverse effects were observed in any of the reproductive or developmental parameters or in the F1 pups. The toxicity profile includes symptoms of minimal to moderate hemosiderosis and minimal congestion in the spleens at 100 mg/kg bw/day males and females. The NOAEL for parental toxicity in the F0 generation was considered to be 30 mg/kg bw/day based on histological effects on the spleen. The minor changes observed in male kidneys at 30 mg/kg bw/day was not considered to be significant. The NOAEL for the F1 generation and reproductive toxicity was considered to be >100 mg/kg bw/day based on no effects observed at the highest dose.

Endpoint:
one-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Wasox-MMAC2 undergoes rapid hydrolysis in aqueous to acetone oxime and the corresponding silanols. These silanols undergo continuous condensation reactions to produce higher molecular weight siloxanes which are considered biologically unavailable. Therefore, the observed toxicity is likely due to the acetone oxime and their values are comparable. The source substance MIBKO, shares the same functional groups with the target substance hydrolysis product acetone oxime, and has comparable values for the relevant molecular properties and both are expected to be toxicologically equivalent. Therefore, the toxicity values of the target substance Wasox-VMAC2 and the source substance MIBKO are comparable.
See attached the reporting format and read-across rationale.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
(parental toxicity)
Effect level:
27.25 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: (Read-across from an analogue for which NOAEL = 30 mg/kg bw/day, based on histological effects on the spleen)
Key result
Dose descriptor:
NOAEL
Remarks:
(reproductive toxicity)
Effect level:
> 90.84 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest dose tested
Remarks on result:
other: (Read-across from an analogue for which NOAEL > 100 mg/kg bw/day, based on no effects at the highest dose tested)
Critical effects observed:
not specified
Key result
Dose descriptor:
NOAEL
Remarks:
(F1 toxicity)
Generation:
F1
Effect level:
> 90.84 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest dose tested
Remarks on result:
other: (Read-across from an analogue for which NOAEL >100 mg/kg bw/day, based on no effects at the highest dose tested)
Key result
Dose descriptor:
NOAEL
Remarks:
(developmental and sexual maturation)
Generation:
F1
Effect level:
> 90.84 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at the highest dose tested
Remarks on result:
other: (Read-across from an analogue for which NOAEL > 100 mg/kg bw/day, based on no effects at the highest dose tested)
Critical effects observed:
not specified
Reproductive effects observed:
not specified
Conclusions:
Based on read-across approach from one-generation reproduction study performed with the analogue methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats, the NOAEL for parental toxicity was considered to be 27.25 mg/kg bw/day (based on histopathological effects on the spleen) and the NOAEL for the F1 generataion and reproductive toxicity was considered to be >90.84 mg/kg bw/day (based on no effects observed on reproduction or developmental parameters).
Executive summary:

A one-generation reproduction study was performed according to OECD guideline 415 with the analogue substance methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats. 28 parental rats per sex and per dose, were given methyl isobutyl ketoxime in water by gavage at doses of 0 (control), 10, 30 and 100 mg/kg bw/day during 10 weeks prior to mating, for up to 2 weeks during mating, or until signs of mating were noted. Males were killed following mating, and females were continuously exposed through gestation and lactation. Parental and litter examinations as well as reproduction observations were performed. All surviving male and female parent rats, were sacrificed for postmortem examinations. All stillborn pups, pups found dead, or pups terminated in a moribund condition and those for which the sexual maturation was observed, were subjected to a throrough necropsy. No adverse effects were observed in any of the reproductive or developmental parameters or in the F1 pups. The toxicity profile includes symptoms of minimal to moderate hemosiderosis and minimal congestion in the spleens at 100 mg/kg bw/day males and females. The NOAEL for parental toxicity in the F0 generation was considered to be 30 mg/kg bw/day based on histological effects on the spleen. The minor changes observed in male kidneys at 30 mg/kg bw/day was not considered to be significant. The NOAEL for the F1 generation and reproductive toxicity was considered to be >100 mg/kg bw/day based on no effects observed at the highest dose. Based on these results, the read-across approach was applied and the NOAEL for P and F1 for the target substance were determined to be 27.25 mg/kg bw/day and >90.84 mg/kg bw/day respectively.

Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
November 5, 2004 - February 23, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Repeated dose toxicity study (28 days, oral) in accordance with OECD 407. GLP study. The study includes the analysis of reproductive organs and tissues.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Fischer 344
Details on species / strain selection:
Fischer, F344/NHsd, SPF
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH, 0-33176 Borchen.
- Age at study initiation: about 7 weeks
- Weight at study initiation: mean males: 152-154 g; mean females: 110-112 g.
- Housing: Group caging (2 or 3 animals of one group and one sex per cage). The first 3 animals of a group and the second 2 animals of a group are housed together. Makroion cages Type IV (33 cm x 55 cm area, 20 cm height). Bedding material: Aspen wood chips.
- Diet (e.g. ad libitum): ad libitum (Altromin 1324). Exception: feed was withdrawn the day before blood sampling and was offered again immediately thereafter.
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): average of 21.9 ºC (continuous control and recording).
- Humidity (%): average of 45.5 % (continuous control and recording).
- Air changes (per hr): 12 per hour
- Photoperiod (hrs dark / hrs light): artificial light from 6 a.m. to 6 p.m.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance was blended with the vehicle (corn oil). Preparations of the test substance were made freshly every day shortly before the administration to the animals. Appropriate preparations were made to allow a uniform dose volume for all groups.

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Altromin 1324 forte, gamma irradiated with 25kGy60Co

VEHICLE
- Justification for use and choice of vehicle (if other than water): as the test substance is known to react immediately with water, corn oil was chosen as solvent.
- Amount of vehicle (if gavage): 5 mL/kg bw
- Purity: the water content of the vehicle was determined by titration.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Determinations of the test substance in some selected preparations for stability, concentration and homogeneity were performed by gas chromatography with flame ionisation detection (GC/FID). The stability, homogeneity and the actual concentration analyses were performed.
Duration of treatment / exposure:
28 days.
Satellite groups were kept after cessation of dosing without a further administration for 15 additional days.
Frequency of treatment:
Once a day
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
63 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals per sex and per dose (control (K), control satellite (KS), low dose (A), mid dose (B), high dose (C) and high dose satellite (CS)).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The doses were derived from the results of the dose range findings study.
Groups of 5 male and 5 female rats were given doses of 100 mg or 316 mg or 1000 mg test substance per kg body weight for 7 consecutive days. Investigations: Body weights, feed consumption, animal observation, gross pathology.
Results: 2 high dosed males died early, with the death related to the test substance. Gross necropsy findings gave some indication for hepatotoxicity. Body weights of the mid and high dosed males were significantly, the corresponding female groups notably, but not significantly, reduced. Feed consumption was reduced in parallel.
Therefore 200 mg test substance per kg body weight, interpolated between low and mid dose of the dose range finding study, are chosen as high dose for the main study. 20 mg test substance per kg, i.e. 10 % of the high dose, is chosen as the low dose, where no toxic effects are anticipated. The mid dose is interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were carefully observed for general signs and the health status once a day and additionally checked once a day for viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Days -3, 4, 11, 18 and 25 (all animals of all groups). Days 32 and 39 (all animals of the satellite groups.
Special emphasis was put on skin, fur, eyes, visible mucous membranes, incisors, secretion and excretion, body odour, autonomous activities (e.g. lacrimation, piloerection, pupillar size, abnormal breathing, and body surface temperature), vocalisation, abnormal locomotion, movements and posture, presence of convulsions or paralysis, stereotypes, bizarre behaviour, visible or palpable tissue masses.

BODY WEIGHT: Yes
- Time schedule for examinations: Day -4 to 31: once a week, all animals. Days 35 and 41: satellite groups.

FOOD EFFICIENCY:
- Food consumption: Determined per cage in weekly intervals in all animals.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was taken on Day 29 from all animals of groups K, A, Band C and of all animals of groups KS and CS on Day 42.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters determined: red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC), mean cell volume (MCV), platelet count (PLT), differential white blood cell count (% of the different cell species), prathrambin time (Quick) as an indicator of blood clotting capacity.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood was taken on Day 29 from all animals of groups K, A, Band C and of all animals of groups KS and CS on Day 42.
- Animals fasted: Yes
- How many animals: 30 animals
- Parameters determined: alanine aminotransferase (ALT, GPT), albumin (ALB), alkaline phosphatase (AP), aspartate aminotransferase (AST, GOT), calcium (Ca), cholesterol (CHOL), creatinine (CREA), gamma-glutamyltransferase (GGT), glucose (GLU), potassium (K), sodium (Na), total protein (TP), urea (UREA)
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes. Necropsy on Day 29 (groups K, A, B, C) and on Day 42 (groups KS and CS).
HISTOPATHOLOGY: Yes: gross lesions, tissue masses or tumours, adrenal glands, aorta*, brain, caecum*, coagulating glands*, colon, duodenum(preparedas "SwissRoll"), epididymides*, eyes*, femur bone with joint*, heart, ileum (prepared as "Swiss Roll"), jejunum (prepared as "Swiss Roll"), kidneys, lacrimal glands (extraorbital)*, liver, lungs, Iymphnodes(mandibular, mesenteric), oesophagus*, ovaries, pancreas *, pituitary*, prostate, rectum*, salivary glands*, seminal vesicles *, sciatic nerve, skeletal muscle (thigh)*, skin and mammary gland*, spinal cord (cervical, thoracic, lumbar), spleen, sternum with bone marrow, stomach, testes, thymus, thyroid and parathyroid glands, trachea, urinary bladder, uterus.
*: not included into routine histopathology of groups K and C.
ORGAN WEIGHTS: Yes: adrenal glands (both together), brain, epididymides (both together), heart, liver, kidneys (both together), spleen, testes (bothtogether), thymus.
Statistics:
Analysis of variance followed by the Scheffé-test, t-test and H-test of Kruskal and Wallis followed by the test of Nemenyi, Chi2-test and Fisher's exact test were used.
Clinical signs:
no effects observed
Description (incidence and severity):
All animals were normal any time. There were no test substance related effects noted.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No test substance related death occurred during the study. All animals survived until their scheduled sacrifice.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
There was no statistically significant difference or dose related trend noted in the body weights of both sexes.
The body weight gain of the mid, the high and the high dosed satellite group males was significantly lower than that of the control group in the first week of dosing (Days 1-8).
The body weight gain of the high dosed satellite group females was significantly higher than that of the corresponding control group in the last week of the recovery period (Days 35-41).
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In the males, the high dosed and the high dosed satellite group had a slightly lower feed consumption during the dosing period, followed by a slightly higher feed consumption in the recovery period, both when compared with the controls. The differences were not markedly pronounced and are given only borderline relevance. There were no note worthy differences or dose related trends noted in the feed consumption of the females. No statistical evaluation of the results is useful due to group caging.
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):
Results included below (see table 1)
All significant differences in haematology are attributed to the test substance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Results included below (see table 2)
All significant differences in clinical biochemistry are attributed to the test substance.
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):
The presence of extramedullary haematopoiesis in the spleens of all high dosed and almost all mid dosed animals of both sexes is related to the test substance. A minor pronounced effect, similar to that mentioned before, is hypercellularity in the bone marrow, noted in a few animals without gaining statistical significance. All other histopathological findings are not related to the test substance and considered as spontaneous alterations without toxicological relevance.
No effects were observed in the ovaries, uterus, prostate, seminal vesicles and testes.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
Dose descriptor:
NOEL
Remarks:
(systemic toxicity)
Effect level:
20 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Dose descriptor:
NOAEL
Remarks:
(reproductive organs)
Effect level:
> 200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (Basis for effect: no effects observed in reproductive organs or tissues)
Critical effects observed:
not specified
Reproductive effects observed:
not specified

No effects were observed in the reproductive organs or tissues up to the highest dose tested (200 mg/kg bw/day).

Conclusions:
The 28day-NOEL of the test substance in rats by oral route was 20 mg/kg bw/day in both sexes.
No effects were observed in the reproductive organs or tissues up to the highest dose tested (200 mg/kg bw/day).
Executive summary:

The Repeated dose 28 -day oral toxicity study in rodents for the test substance Wasox-MMAC2 was performed according to OECD Guideline 407 in F344 rats. The test material was administered blended with corn oil, given orally by gavage to 3 groups of 5 male and 5 female rats, once a day for 28 consecutive days. The dose used were 20, 63, and 200 mg/kg/day (groups A, B and C). A negative control group (group C, treated with the vehicle) was included. In addition, 2 groups of 5 males and 5 females each (i.e. one high dose satellite group CS and one control satellite group KS) were treated in the same way as their corresponding groups, but were kept for further 14 days without test substance administration in an attempt to observe the reversibility or persistence of test substance induced lesions. Clinical signs, functional observations, body weights, feed consumption, haematology, clinical biochemistry, necropsy with gross pathological examination, organ weights and histopathological examinations were performed within the study. One major effect of Wasox MMAC2 can be derived from several results of the present study. The test substance causes damage to mature erythrocytes in the peripheral blood, resulting in alterations in virtually all erythrocyte-related parameters at the haematological examination. In parallel to this damage there is a marked repair by ongoing by an increased new production of young erythrocytes, notable in an increased proportion of polychromatic erythrocytes in the peripheral blood and also in the presence of haematopoiesis in the spleen, inducing even a significant weight increase of this organ, and - less pronounced - a hypercellularity of the bone marrow. The repair was efficient enough to allow the animals to live without physical behavioural abnormalities or body weight effects. There was no indication for a negative effect to the blood cell production in the bone marrow found. A second - minor expressed - effect may be derived from the clinical biochemistry data. Lowered plasma albumin and total protein, lowered glucose (males) and elevated GGT values (females) and a slightly increased liver weight may be due to a borderline hepatic involvement. All indications for hepatotoxicity are only poorly pronounced. Some haematological changes in neutrophil and Iymphocyte counts remain without a clear interpretation, but may be secondary to the erythrocyte alterations. Effects were similarly noted in males and females at the same doses, no sex difference in the response to the test substance can be derived from the results. Effects were only partly reversible during the recovery period, though there was a clear tendency towards a return to normal. The NOEL of Wasox-MMAC2 was at 20 mg per kg body weight and day in both sexes. No effects were observed in the reproductive organs or tissues up to the highest dose tested (200 mg/kg bw/day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
90.84 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
One-generation toxicity study is available on analogue substances with a Klimish score = 2 since read-across was applied. Moreover, as supporting study, one repeated dose toxicity test is in place (Klimish score = 2) where effects on reproductive organs were analised. The overall quality of the database was determined as appropriate for assessment.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Key study Read-across from experimental data on analogue substance MIBKO:

A one-generation reproduction study was performed according to OECD guideline 415 with methyl isobutyl ketoxime (MIBKO) in Sprague-Dawley rats. No adverse effects were observed in any of the reproductive or developmental parameters or in the F1 pups. The toxicity profile includes symptoms of minimal to moderate hemosiderosis and minimal congestion in the spleens at 100 mg/kg bw/day males and females. The NOAEL for parental toxicity in the F0 generation was considered to be 30 mg/kg bw/day based on histological effects on the spleen. The minor changes observed in male kidneys at 30 mg/kg bw/day was not considered to be significant. The NOAEL for the F1 generation and reproductive toxicity was considered to be >100 mg/kg bw/day based on no effects observed at the highest dose. Based on these results the read-across was applied and the NOAEL of Wasox-MMAC2 for parental toxicity and the NOAEL for F1 generation and toxicity to reproduction were estimated to be 27.25 and >90.84 mg/kg bw/day respectively.

Supporting study: Experimental data on Wasox-MMAC2:

A 28 days oral repeated dose toxicity test was performed on Wasox-MMAC2 according to OECD Guideline 407 and GLP. The NOEL was determined to be 20 mg/kg bw/day in rats since effects on erythrocyte related parameters, liver and spleen weight changes, haematopoiesis in the spleen and hypercellularity in the bone marrow were observed. Based on these results, the read-across was applied and the 28 days NOEL for oral exposure in rats was estimated to be 20 mg/kg bw/day under the test conditions. No effects were observed in the reproductive organs or tissues up to the highest dose tested (200 mg/kg bw/day).

Justification for selection of Effect on fertility via oral route:

One-generation study is available.

Justification for classification or non-classification

Based on the available information on toxicity to reproduction toxicity, the substance is not classified in accordance with CLP Regulation (EC) No 1272/2008.

Additional information