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Description of key information

In an oral OECD 422 study with rats, the NOAEL was determined to be at least 1000 mg/kg bw/day, based on no adverse effects seen at this dose level.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 November 2014 - 02 February 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
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:
(1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) (2000)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OECD 421, Reproduction/Developmental Toxicity Screening Test (July 1995)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents (July 2000)
Deviations:
no
GLP compliance:
yes
Limit test:
no
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
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 10 weeks (males) or 11 weeks (females)
- Weight at study initiation: 294-330 g (males) or 186-223 g (females)
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS (set conditions)
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
Temporary deviations from the daily mean relative humidity occurred in the room in which selected animals were kept temporarily for motor activity measurents (daily mean values were not lower than 37%). As laboratory historical data do not indicate an effect of the deviations, the study integrity was not affected.
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

- Experimental starting date: 11 November 2014 (randomization dose range finding study)
- Start treatment: 8 December 2014
- Experimental completion date: 2 February 2015 (end of in-life phase)
Route of administration:
oral: gavage
Vehicle:
other: 1% aqueous carboxymethyl cellulose
Details on oral exposure:
- Method of formulation: Formulations (w/v) were prepared daily prior to dosing and homogenized to a visually acceptable level. No correction was made for the purity/composition of the test substance. Initially (day 1-3), formulations were prepared within 6 hours prior to dosing. From day 4 onwards, the time between preparation of the test substance formulations and dosing was kept as brief as possible. The actual time between preparation and dosing of the test substance formulations was maximally about 4 hours on days 1-3 and maximally about 2 hours thereafter.
- Storage conditions of formulations: At room temperature
- Justification for use and choice of vehicle: The vehicle was choosen based on trial formulations performed at WIL Research Europe.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Analyses were conducted on a single occasion during the treatment phase (10 December 2014), according to a validated method (WIL Research Project 506993, see IUCLID section 8).
- Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
- 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 ≤ 10%.
- Stability of the test substance in the formulations was not determined because this was not possible with the analytical method used (inductively coupled plasma – mass spectrometry (ICPS-MS) based on strontium in the test substance). The test substance is assumed to be stable according to its structure.
Duration of treatment / exposure:
Males were exposed for 32 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females were exposed for 42-56 days, i.e. during 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).
Frequency of treatment:
Once daily, 7 days/week
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
Basis:
actual ingested
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 (WIL Research Project 507000). In this DRF study, 3 females were dosed with either 500 or 1000 mg/kg bw/day. No signs of toxicity were noted in this study.
- Selection of animals for selected measurements: 5 animals/sex/group were selected for functional observations, locomotor activity, clininal pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live pups were selected.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS:
Yes
- Time schedule: At least once daily from start of treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals during the treatment period, this was also performed outside the home cage in a standard arena.

BODY WEIGHT:
Yes
-Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on days 1 and 4.

FOOD CONSUMPTION:
Yes
- Weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 postcoitum and on days 1 and 4 of lactation.

FOOD EFFICIENCY:
Yes. (average food consumption [per animal per day]/average body weight per cage)x1000

WATER CONSUMPTION : No.
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Anaesthetic used for blood collection: Yes (iso-flurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY:
Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination.
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS: No

FUNCTIONAL OBSERVATIONS:
Yes
- Time schedule for examinations: The selected males were tested during week 4 of treatment and the selected females were tested towards the end of the scheduled lactation period (all before blood sampling).
- Dose groups that were examined: 5 animals/sex/group
- Battery of functions tested: Hearing ability, pupillary reflex and static righting reflex; Fore- and hind-limb grip strength; Locomotor activity.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated. Two females dosed at 300 mg/kg bw/ day and one female dosed at 1000 mg/kg bw/ day (all three were found to be not pregnant) were dosed on day of necropsy.

- All surviving animals
- All animals and all animals that died spontaneously or were killed in extremis

ORGAN WEIGHTS
- Selected 5 animals/sex/group: all organs as specified in the test guidelines

- All remaining males:
Epididymides and testes

HISTOPATHOLOGY: Yes
All organs and tissues as specified in the test guidelines

No organ weights and terminal body weight were determined at necropsy of one male dosed at 1000 mg/kg bw/ day. This rat died at blood sampling prior to necropsy. Full histopathological examination of this male was performed. The coagulation glands from one male dosed at 300 mg/kg bw/ day were not available for histopathology.
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The 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. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
No mortality was observed, except for one male dosed at 1000 mg/ kg bw/ day, which died at blood sampling (death not substance-related).

CLINICAL SIGNS
No clinical signs of toxicity were noted in any of the rats.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were not affected by the treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted. In males at 1000 mg/kg bw/ day lower cumulative body weight gain was noted during the mating period. The differences from control were statistically significant at days 1 and 15 of this period (-4 g and - 5 g resp.). As mean body weights of males at 1000 mg/kg bw/ day did not differ significantly from those of controls, the differences in body weight gain were considered not to be toxicologically relevant. Mean terminal body weight of females at 1000 mg/kg bw/ day was lower than that of controls (relative difference: 10%). At day 4 of the lactation period, females at 1000 mg/kg bw/ day and controls had similar mean body weights based on 7 and 10 females, respectively. The terminal body weights were determined at days 5, 6 or 7 of lactation from a selection of these females (5/ group) after overnight fasting. The difference in mean terminal body weights was likely to be the result, at least in part, of the selection and fasting of the females. Therefore, this difference was considered not to reflect treatment-related toxicity.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight showed no toxicologically or statistically significant differences between treated and control animals.

HAEMATOLOGY
No toxicologically relevant changes were observed in males. Females at 1000 mg/kg bw/ day had statistically significantly higher values for activated partial thromboplastin time (+ 3.8s). As this value falls within historical range, this increase was not found to be toxicologically relevant. No other toxicologically relevant changes were observed.

CLINICAL BIOCHEMISTRY
Males at 1000 mg/kg bw/ day had higher blood values for alkaline phosphatase (ALP) and inorganic phosphate (+49% and + 23% resp.), but the values of these parameters at 1000 mg/kg bw/ day remained in the normal range for male rats of this age and strain. As this effect was not accompanied by other relevant changes in other parameters or histopaythological changes, therefore these differences in ALP and inorganic phosphate were considered not to be adverse. Furthermore, at all dose levels total protein levels and albumin levels were decreased. In absence of dose-relationship and given the fact that changes were less than 10% compared to control values, the changes were not found to be adverse. In females, no toxicological relevant changes in clinical biochemistry were noted.

MACROSCOPIC EXAMINATION
There were no test item-related gross observations. Incidental findings in males consisted of several reddish foci in thymus of two rats dosed at 300 mg/ kg bw/ day and one rat with its right medial lobe grown together with its diaphragm. At 1000 mg/kg bw/ day, one animal was found to have an enlarged right side auricle, several reddish foci in the lung and several observations in liver (diaphragmatic hernia, enlarged and discoloured right medial lobe (dark red)). Several occasional observations were seen (irregular surface of glandular mucosa of the stomach and enlarged mandibular lymph nodes in one female each dosed at 300 mg/kg bw/ day, fluid in uterus (2 rats at 300 mg/kg bw/ day, 1 rat at 1000 mg/kg bw), stomach irregularities (one with irregular surface of the glandular mucosa and one with dark red foci, at 1000 mg/ kg bw/ day), reddish focus on clitoral gland and an enlarged spleen (resp 2 and 1 rats at 1000 mg/ kg bw/ day), but as there was no dose-related increase in incidence and/or severity, they were found not to be related to exposure to L 130.

ORGAN WEIGHTS
Males dosed at 1000 mg/kg bw/ day had statistically significantly lower thymus weights (absolute: -17%, -14% and - 29% and relative: -14%, -12% and -26%, for groups dosed at 100, 300 and 1000 mg/kg bw/ day, respectively). The lower thymus weight was not associated with histological changes and within the historical control range of the laboratory. Therefore, this finding was considered not to be toxicologically relevant. Absolute and relative spleen weight was decreased related to control in all groups (absolute: -10%, -4% and - 10% and relative: -7%, -1% and -15%, for groups dosed at 100, 300 and 1000 mg/kg bw/ day, respectively). In absence of histopathological effects this effect was found to be not adverse.
Females at 100 and 1000 mg/kg bw/ day had higher heart to body weight ratios than controls. These differences showed no dose-related response and were considered to be due to incidental intergroup differences in terminal body weights of the selected females. Absolute and relative mean liver weights of females were decreased compared to the control (absolute: -20%, +8%, -22% and relative: -14%, +16% and -13%, for females dosed at 100, 300 and 1000 mg/kg bw/ day, respectively). This effect can be accounted for by one control female with a very high liver weight (12.2 g versus average of other control animals of 7.9 g) and was thus not found to be toxicologically relevant. Absolute and relative thymus weight was decreased for females dosed at 100 and 1000 mg/kg bw/ day (absolute: -20% and -22%, relative: -14% and -13%, respectively) In absence of a dose-relationship and given the fact that one female was found with a relatively high thymus weight in the control group influencing the results, this effect was found to be not toxicologically relevant.
Furthermore, uterus weight (absolute and relative) was increased in all exposed groups (absolute: +11%, +2.7% and +14%, relative: +19%, +10% and +27% for females dosed at 100, 300 and 1000 mg/ kg bw/ day, respectively). In both the groups exposed to 100 and to 1000 mg/ kg bw/ day the high average is related to presence of one female with a relatively higher uterine weight. Therefore, this observation is not found to be adverse. The other organ weights and organ to body weight of treated animals were similar to those of control animals.

MICROSCOPIC EXAMINATION
There were no test item-related histologic changes (spermatogenic staging profiles were normal for all males examined).
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 were observed at the highest dose level tested.
Critical effects observed:
not specified

The concentrations analyzed in the formulations of the three test groups were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). A small response at the m/z of the relevant element of the test substance was observed in the formulation of the control group. Since the maximum contribution was 1.1%, it was considered not to have a significant impact on the results. The formulations of groups dosed at 100 and 1000 mg/kg bw/ day were homogeneous (i.e. coefficient of variation ≤ 10%).

Conclusions:
In an oral OECD422 study with rats, the NOAEL was determined to be at least 1000 mg/kg bw/day, based on no adverse effects seen at this dose level.
Executive summary:

An oral OECD422 study was performed with rats according to OECD/ EC guidelines and GLP principles. Male and female rats were exposed to L130 via oral gavage to 0, 100, 300 or 1000 mg/kg bw/ day for 32 days (males) or 42-56 days (females). Accuracy and homogeneity of formulations were confirmed by analyses. No substance-related mortality occurred during the study. No clinical signs of toxicity were noted in any of the rats. No functional changes were observed related to the treatment. No toxicologically relevant changes in body weights and body weight gain were noted. No differences in food consumption were observed between treated and control animals. In males, at all dose levels total protein levels and albumin levels were decreased. In absence of dose-relationship and given the fact that changes were less than 10% compared to control values, the changes were not found to be adverse. No toxicologically relevant changes were measured in blood parameters of males and females, all observed changes remained within historical ranges of this strain. There were no test item-related gross observations. Thymus weights of both males and females were decreased (for males: absolute and relative at all doses; for females: absolute and relative 100 and 1000 mg/kg bw/ day. The severity of the decrease of thymus weights was not dose-related, not associated with histological changes and within the historical control range of the laboratory. Therefore, this finding was considered not to be toxicologically relevant. Absolute and relative spleen weight in males was decreased related to control in all groups. In absence of histopathological effects this effect was found to be not adverse. There were no test item-related histologic changes.

In conclusion, the NOAEL of L 130 was determined to be at least 1000 mg/kg bw/day, based on no adverse effects seen at this dose level.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The study was performed according to OECD/ EC guidelines and GLP principles (Klimisch 1).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

An oral OECD422 study was performed with rats according to OECD/ EC guidelines and GLP principles. Male and female rats were exposed to L130 via oral gavage to 0, 100, 300 or 1000 mg/kg bw/ day for 32 days (males) or 42-56 days (females). Accuracy and homogeneity of formulations were confirmed by analyses. No substance-related mortality occurred during the study. No clinical signs of toxicity were noted in any of the rats. No functional changes were observed related to the treatment. No toxicologically relevant changes in body weights and body weight gain were noted. No differences in food consumption were observed between treated and control animals. In males, at all dose levels total protein levels and albumin levels were decreased. In absence of dose-relationship and given the fact that changes were less than 10% compared to control values, the changes were not found to be adverse. No toxicologically relevant changes were measured in blood parameters of males and females, all observed changes remained within historical ranges of this strain. There were no test item-related gross observations. Thymus weights of both males and females were decreased (for males: absolute and relative at all doses; for females: absolute and relative 100 and 1000 mg/kg bw/ day. The severity of the decrease of thymus weights was not dose-related, not associated with histological changes and within the historical control range of the laboratory. Therefore, this finding was considered not to be toxicologically relevant. Absolute and relative spleen weight in males was decreased related to control in all groups. In absence of histopathological effects this effect was found to be not adverse. There were no test item-related histologic changes.

In conclusion, the NOAEL of L130 was determined to be at least 1000 mg/kg bw/day, based on no adverse effects seen at this dose level.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
One study available.

Justification for classification or non-classification

Based on the results of a combined repeated dose and reproduction / developmental screening study, L130 is not classified for repeated dose toxicity according to the CLP Regulation.