Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

A 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guidelines and in accordance with GLP principles is available. No adverse effects were observed up to and including 1000 mg/kg bw/day, the highest dose tested.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3550
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3050
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: yes, the substance is not expected to disintegrate in the vehicle and under the test conditions used in the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogenized to a visual acceptable level
Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10-12 weeks
- Weight at study initiation: males 281-318 grams, females 195-230 grams
- Fasting period before study: no
- Housing: in Macrolon plastic cages; premating in groups of 5 animals/sex/cage; females were caged together with males on a one-to-one basis for mating; post-mating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed; pups were kept with the dam until termination. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: free acess to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiӓten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: at least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting materi
al evaluation for contaminants and/or nutrients was performed according to facility standard procedures.
There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 January 2016 To: 01 March 2016
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/v) were prepared daily within 6 hours prior to dosing and were homogenized to a visual acceptable level.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days was allowed for mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post coitum
- After successful mating each pregnant female was caged: individually in plastic cages
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase. On the first occasion samples from dosing formulations were analyzed for homogeneity (low and high concentrations) and accuracy
of preparation (all concentrations, including vehicle control). Samples from mid and high concentration dosing formulations prepared on the second occasion were additionally analysed for homogeneity and
accuracy.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was
equal or below 10%. Analysis of stability of the test substance under test conditions was not performed. The substance is a compact mixed metal oxide produced in a high temperature calcination process and is not expected to disintegrate in the chosen vehicle and under the test conditions used in this study, based on the following reasons:
-the substance is considered insoluble in water
-experiences made during the development of an ICP-MS method for analysis of the test substance in test media, i.e. very harsh conditions (mixture of strong mineral acids in combination with microwave di
gestion) are needed to decompose the substance into its metal ions
-due to the inorganic nature of the substance, stability measurements, based on an element in the substance, are not meaningful.
Duration of treatment / exposure:
males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
females: 42-54 days (2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation, up to the day prior to scheduled necropsy)
Pups were not dosed directly.
Frequency of treatment:
once daily for 7 days per week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-day dose range finding study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighted on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 4 of treatment, females during days 3-6 of lactation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity/ hearing ability / grip strength / motor activity / pupillary reflex
Oestrous cyclicity (parental animals):
no
Sperm parameters (parental animals):
testis weight, epididymis weight, weight of seminal vesicles including coagulation glands, prostate weight, staging of spermatogenesis
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after 14 days of mating period
- Maternal animals: All surviving animals after a 4-day lactation period

GROSS NECROPSY
- Gross necropsy according to guideline

HISTOPATHOLOGY / ORGAN WEIGHTS
According to guideline.
Postmortem examinations (offspring):
Pups surviving to planned termination were killed by decapitation on Days 5-7 of lactation. All pups were sexed and descriptions of all external abnormalities were recorded. The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.

Statistics:
Statistical methods used to analyze the data:
-Dunnett-test (many-to-one t-test) based on a pooled variance estimatewas applied for the comparison of the treated groups and the control groups for each sex if the variables could be assumed to follow normal distribution.
-Steel-test (many-to-one rank test) if the data could not be assumed to follow a normal distribution.
-Fisher Exact-test was applied to frequency data.
Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Reproductive indices:
Mating index (%) = (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
Offspring viability indices:
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 = (Number of dead pups before planned necropsy / Number of live pups at First Litter Check) x 100
Viability index = (Number of live pups before planned necropsy / number of pups born alive) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hunched posture and/or piloerection were observed on two separate occasions in two control females. At the incidence observed and because of their short-term presence in controls only, these signs were considered of no toxicological relevance.
Mortality:
no mortality observed
Description (incidence):
No mortality occured during the study period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of the treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 1000 mg/kg bw/day treated females (statistically significant) increase in number of red blood cells and increased values of Hb and Ht were observed when compared to controls. In these females MCH, MCV and MCHC values (red cell derived parameters) were similar in comparison with the other dose groups including controls. The statistically significant difference apparent for the number of red blood cells in 300 mg/kg bw/day treated females was considered to be the result of a relatively low variation in the individual values within the dose group and to have occurred by change.
In males treated at 300 mg/kg bw/day a lower Hb value was observed, and in males from the 300 and 1000 mg/kg bw/day groups (stat sign) higher APTT values were observed compared to controls.
In females, in dose group 100 mg/kg bw/day a (stat sign) higher relative lymphocyte number and lower number of neutrophilswere observed. Lower RDW values were observed in females of all treated
groups; lower MCH value was observed at 100 mg/kg bw/day and increased PT at 1000 mg/kg bw/day (achieving stat sign in most cases when compared to controls).
These changes were considered to be of no toxicological relevance as they were the result of relatively high control values (RWD) or occurred in the absence of treatment-related distribution and remained within the range considered normal for rats of this age and strain.
In a single female in dose group 300 mg/kg bw/day a low number of white blood cells was observed together with an increased number of neutrophils and decreased number of lymphocytes. This observation is often related to a non-specific stress response, which may be supported by the observations in the thymus. Since this observation was limited to a single female only and showed no dose effect relationship these findings were considered of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean value for total bilirubin in females dosed 100 mg/kg bw/day (stat sign when compared to controls) was considered to have occured by chance and to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The foreleg grip strength in females showed a relatively large difference between the treated groups and controls but in teh absence of a dose-response relationship and absence of corroborative effect in the hindlegs this finding was considered of no toxicological relevance. Moreover, all fore leg grip strength values were within the normal limits. The variation in motor activity did not indicate a relation with treatment, as all groups showed the a similar habituation profile.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including 1000 mg/kg bw/day (highest dose tested)
Key result
Critical effects observed:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
no effects observed during lactation
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including 1000 mg/kg bw/day, the highest dose tested
Reproductive effects observed:
no

In a 10 -Day range-finding study, 3 females/group were dosed with 500 or 1000 mg/kg bw/day. Mortality, clinical signs , body weights and food consumption,were recorded At necropsy all animals were subjected to an external, thoracic and abdominal examination, and organ weights of liver and kidney were measured.

In this range-finding study only clinical signs (temporary hunched posture and/or piloerection mainly between 1 to 3 hours after dosing) were observed. Based on these results, the dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.

Conclusions:
Treatment with L 125 PLUS by oral gavage in male and female rats did not result in parental, reproductive and developmental adverse effects up to and including 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

L 125 was tested in a 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422. L 125 PLUS was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for two weeks prior to mating, during mating, and up to termination (29 days). The females were exposed for two weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously.

No treatment related adverse effects were observed in the parental males and females up to and including the highest dose level tested (1000 mg/kg bw/day) as evidenced by the absence of signs of toxicity, haematology or adverse changes in clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination. Based on these results a NOAEL (parental) of at least 1000 mg/kg bw/day was derived. No reproductive or developmental toxicity was observed up to and including 1000 mg/kg bw/day (the highest dose tested), and based on these results, NOAELs for both reproduction and develpmental toxicity are at least 1000 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The substance was tested in a 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422 and was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for two weeks prior to mating, during mating, and up to termination (29 days). The females were exposed for two weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously. No treatment related adverse effects were observed in the parental males and females up to and including the highest dose level tested (1000 mg/kg bw/day). Based on these results a NOAEL (parental) of at least 1000 mg/kg bw/day was derived.

No reproduction toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, and numbers of corpora lutea and implantation sites, spermatogenic profiling and histopathological examination of reproductive organs). Based on these results a NOAEL (reproduction) of at least 1000 mg/kg bw/day was derived.

Effects on developmental toxicity

Description of key information

A 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guidelines and in accordance with GLP principles is available. No adverse effects were observed up to and including 1000 mg/kg bw/day, the highest dose tested.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 2015 - March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
March 1996
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3650
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
July 1995
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
October 2008
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3550
Version / remarks:
July 2000
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3050
Version / remarks:
July 2000
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: yes, the substance is not expected to disintegrate in the vehicle and under the test conditions used in the study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: homogenized to a visual acceptable level
Species:
rat
Strain:
other: Crl:WI(Han)
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10-11 weeks
- Weight at study initiation: males 281-318 grams, females 195-230 grams
- Fasting period before study: no
- Housing: in Macrolon plastic cages; premating in groups of 5 animals/sex/cage; females were caged together with males on a one-to-one basis for mating; post-mating males were housed in their home cage with a maximum of 5 animals/cage, females were individually housed; pups were kept with the dam until termination. Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied.
- Diet: free acess to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiӓten GmbH, Soest, Germany)
- Water: Free access to tap-water
- Acclimation period: at least 5 days prior to start of treatment

DETAILS OF FOOD AND WATER QUALITY: Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 January 2016 To: 01 March 2016
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/v) were prepared daily within 6 hours prior to dosing and were homogenized to a visual acceptable level.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were conducted on two occasions during the treatment phase. On the first occasion samples from dosing formulations were analyzed for homogeneity (low and high concentrations) and accuracy of preparation (all concentrations, including vehicle control). Samples from mid and high concentration
dosing formulations prepared on the second occasion were additionally analysed for homogeneity and accuracy.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was equal or below 10%. Analysis of stability of the test substance under test conditions was not performed.
The substance is a compact mixed metal oxide produced in a high temperature calcination process and is not expected to disintegrate in the chosen vehicle and under the test conditions used in this study, based on the following reasons:
-the substance is considered insoluble in water
-experiences made during the development of an ICP-MS method for analysis of the test substance in test media, i.e. very harsh conditions (mixture of strong mineral acids in combination with microwave digestion) are needed to decompose the substance into its metal ions
-due to the inorganic nature of the substance, stability measurements, based on an element in the substance, are not meaningful.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: a maximum of 14 days was allowed for mating
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 post coitum
- After successful mating each pregnant female was caged: individually in plastic cages
- Any other deviations from standard protocol: no
Duration of treatment / exposure:
males: 29 days (2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy)
females: 42-54 days (2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation, up to the day prior to scheduled necropsy)
Pups were not dosed directly but could have potentially been exposed to the test item in utero, via maternal milk or from exposure to maternal urine/faeces.
Frequency of treatment:
once daily for 7 days per week
Duration of test:
until at least 4 days of lactation of the pups
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels were based on results of a 10-day dose range finding study.
Maternal examinations:
Parental animals: Observations and examinations
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of exposure and weekly thereafter. Mated females were weighted on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/
kg body weight/day: Yes
FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Animals fasted: Yes (o/n)
- How many animals: all parental animals
- Parameters according to guidelines were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: males during week 4 of treatment, females during days 3-6 of lactation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / pupillary reflex

Estrous cyclicity (parental animals)
no

Sperm parameters (parental animals)
Parameters examined in male parental generation: testis weight, epididymis weight, weight of seminal vesicles including coagulation glands, prostate weight, staging of spermatogenesis
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No
Fetal examinations:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities. Possible cause of death was determined for pups born or found dead.

Statistics:
Statistical methods used to analyze the data:
-Dunnett-test (many-to-one t-test) based on a pooled variance estimatewas applied for the comparison of the treated groups and the control groups for each sex if the variables could be assumed to follow normal distribution.
-Steel-test (many-to-one rank test) if the data could not be assumed to follow a normal distribution.
-Fisher Exact-test was applied to frequency data.
Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p<0.05 was accepted as the lowest level of significance.
Indices:
Offspring viability indices
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 = (Number of dead pups before planned necropsy / Number of live pups atFirst Litter Check) x 100
Viability index = (Number of live pups before planned necropsy / number of pups born alive) x 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Hunched posture and/or piloerection were observed on two separate occasions in two control females. At the incidence observed and because of their short-term presence in controls only, these signs were considered of no toxicological relevance.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of the treated animals remained in the same range as controls over the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before and after allowance for body weight was similar between treated and control animals.
Food efficiency:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In 1000 mg/kg bw/day treated females (statistically significant) increase in number of red blood cells and increased values of Hb and Ht were observed when compared to controls. In these females MCH, MCV and MCHC values (red cell derived parameters) were similar in comparison with the other dose groups
including controls. The statistically significant difference apparent for the number of red blood cells in 300 mg/kg bw/day treated females was considered to be the result of a relatively low variation in the individual values within the dose group and to have occurred by change. In males treated at 300 mg/kg bw/day a lower Hb value was observed, and in males from the 300 and 1000 mg/kg bw/day groups (stat sign) higher APTT values were observed compared to controls. In females, in dose group 100 mg/kg bw/day a (stat sign) higher relative lymphocyte number and lower number of neutrophilswere observed. Lower RDW values were observed in females of all treated groups; lower MCH value was observed at 100 mg/kg bw/day and increased PT at 1000 mg/kg bw/day (achieving stat sign in most cases when compared to controls). These changes were considered to be of no toxicological relevance as they were the result of relatively high control values (RWD) or occurred in the absence of treatment-related distribution and remained within the range considered normal for rats of this age and strain. In a single female in dose group 300 mg/kg bw/day a low number of white blood cells was observed together with an increased number of neutrophils and decreased number of lymphocytes. This observation is often related to a non-specific stress response, which may be supported by the observations in the thymus. Since this observation was limited to a single female only and showed no dose effect relationship these findings were considered of no toxicological relevance.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Mean value for total bilirubin in females dosed 100 mg/kg bw/day (stat sign when compared to controls) was considered to have occurred by chance and to be of no toxicological relevance.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The foreleg grip strength in females showed a relatively large difference between the treated groups and controls but in the absence of a dose-response relationship and absence of corroborative effect in the hindlegs this finding was considered of no toxicological relevance. Moreover, all fore leg grip strength values were within the normal limits. The variation in motor activity did not indicate a relation with treatment, as all groups showed a similar habituation profile.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Thymus weight (abs and rel) was slightly (stat sign) increased in high dose females compared to controls. Slightly higher (stat sign) relative kidney weights were noted in mid and high dose males compared to controls. The changes observed in thymus weights in females and kidney weights in males are within the the normal limits for rats of this age and strain and therefore not considered to be toxicologically relevant.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
not examined
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Changes in number of pregnant:
no effects observed
Description (incidence and severity):
The precoital time showed that confirmation of the first mated females in the control, low and mid dose groups were detected on day 1 of the pairing period, whereas this was on day 2 for the high dose females. The distribution of mated high dose females over the pairing period, however, was comparable to that in the other dose groups, resulting in a similar median precoital time, but slightly higher mean precoital time in the high dose females when compared to the other groups. As at least 9 out of 10 females per group mated within 5 days of pairing, precoital time was considered unaffected by treatment in all groups.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: no adverse effects observed up to and including 1000 mg/kg bw, the highest dose tested
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
At first litter check, one pup from a control litter and two pups from a litter at 300 mg/kg bw/day were found dead. During lactation, one pup from a control litter, two pups from a litter at 300 mg/kg bw/day and one pup from a litter at 1000 mg/kg bw/day went missing, one pup from a control litter was found dead and one pup from a litter at 300 mg/kg bw/day had to be sacrificed for humane reasons. The missing pups were most likely cannabilised. No toxicological relevance was attributed to these dead and missing pups, since the incidences did not show a dose-related trend and remained within the range considered normal in reproduction studies with rats of this age and strain.
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed up to and including 1000 mg/kg bw/day, the highest dose tested
Developmental effects observed:
no

In a 10 -Day range-finding study, 3 females/group were dosed with 500 or 1000 mg/kg bw/day. Mortality, clinical signs , body weights and food consumption,were recorded At necropsy all animals were subjected to an external, thoracic and abdominal examination, and organ weights of liver and kidney were measured.

In this range-finding study only clinical signs (temporary hunched posture and/or piloerection mainly between 1 to 3 hours after dosing) were observed. Based on these results, the dose levels for the main study were 100, 300 and 1000 mg/kg bw/day.

Conclusions:
Treatment with L 125 PLUS by oral gavage in male and female rats did not result in parental, reproductive and developmental adverse effects up to and including 1000 mg/kg bw/day, the highest dose tested.
Executive summary:

L 125 was tested in a 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422. L 125 PLUS was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for two weeks prior to mating, during mating, and up to termination (29 days). The females were exposed for two weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously. No treatment related adverse effects were observed in the parental males and females up to and including the highest dose level tested (1000 mg/kg bw/day) as evidenced by the absence of signs of toxicity or adverse changes in clinical appearance, haematology, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights and microscopic examination. Based on these results a NOAEL (parental) of at least 1000 mg/kg bw/day was derived. No reproductive or developmental toxicity was observed up to and including 1000 mg/kg bw/day (the highest dose tested), and based on these results, NOAELs for both reproduction and develpmental toxicity are at least 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
good
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

The substance was tested in a 28-Day repeated dose toxicity study with the reproduction/developmental toxicity screening test according to OECD guideline 422 and was administered by daily oral gavage to male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg bw/day. Males were exposed for two weeks prior to mating, during mating, and up to termination (29 days). The females were exposed for two weeks prior to mating, during mating, during post-coitum, and at least 4 days of lactation (for 42 -54 days). Formulation analysis showed that the formulations were prepared accurately and homogenously. No treatment related adverse effects were observed in the parental males and females up to and including the highest dose level tested (1000 mg/kg bw/day). Based on these results a NOAEL (parental) of at least 1000 mg/kg bw/day was derived.

No developmental toxicity was observed up to and including the highest dose level tested (1000 mg/kg bw/day). No treatment-related changes were noted in any of the developmental parameters investigated in this study (i.e. gestation index and duration, parturition, maternal care and early postnatal pup development consisting of mortality, clinical signs, body weight and macroscopy). Based on these results a NOAEL (development) of at least 1000 mg/kg bw/day was derived.

Justification for classification or non-classification

In a reliable 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were observed up to and including 1000 mg/kg bw/day, the highest dose tested. According to the criteria outlined in the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 (CLP Regulation), the substance does not need to be classified for reproduction toxicity or developmental toxicity.