Registration Dossier

Administrative data

short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 August 2016 - 22 November 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to guideline
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
Limit test:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
Appearance: Colourless to light yellow viscous liquid
Batch: 16020201
Purity/Composition: 99.56%
Test item storage: At room temperature
Stable under storage conditions until: 21 March 2021 (expiry date)

Test animals

Details on species / strain selection:
Crl:WI(Han) (outbred, SPF-Quality). This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Males: approximately 11 weeks. Females: approximately 13 weeks.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

Administration / exposure

Route of administration:
oral: gavage
corn oil
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
Analyses were conducted on a single occasion during the treatment phase (10 October 2016) according to a validated method (Test Facility Study No. 513599).
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 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Duration of treatment / exposure:
Males were dosed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy. Females that delivered were dosed for 50-55 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were treated for 38 or 42 days.
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces.

Frequency of treatment:
Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Positive control:


Observations and examinations performed and frequency:
Mortality / viability, clinical signs (daily), functional observations and locomotor activity (end of treatment), body weight and food consumption (at least at weekly intervals), estrous cycle determination (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy), clinical pathology (end of treatment), measurement of thyroid hormone T4 (F0-males at the end of treatment, PND 13-15 pups), macroscopy at termination, organ weights and histopathology on a selection of tissues.
In addition, the following reproduction/developmental parameters were determined: mating, fertility and conception indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weights, anogenital distance, areola/nipple retention and macroscopy).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see list)

HISTOPATHOLOGY: Yes - all tissues subjected to gross pathalogy also underwent histopathological examination.
The following statistical methods were used to analyse 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.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were noted during daily clinical observations or during weekly arena observations. Clinical findings noted incidentally occurred within the range of background findings to be expected for rats of this strain and age which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.
no mortality observed
Description (incidence):
One female (no. 64) at 300 mg/kg was sacrificed on PND 1 because of total litter loss (n=1).
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of 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 or 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
Description (incidence and severity):
Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
Haematological parameters were not affected by treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry parameters were not affected by treatment. The statistically significant variations noted in potassium (females) were unrelated to treatment because they occurred in the absence of a dose-related response. A few outlying values were noted in treated females (high urea and creatinine at 1000 mg/kg; high bile acids at 300 and 1000 mg/kg). As these outlying values occurred in only a single animal of a group and in the absence of corroborative changes indicative of renal or hepatic toxicity, they were considered not to reflect adverse effects of the test item.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment. The statistically significant differences noted in hind limb grip strength (higher in males at 100 mg/kg and in females at 300 mg/kg) occurred in the absence of a dose-related response and were therefore considered to be unrelated to treatment. The variation in motor activity did not indicate a relation with treatment. All groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. Mean values for total movements and/or ambulations were lower in males at 300 mg/kg and higher in females at 300 and 1000 mg/kg. The differences from controls were not statistically significant and showed no (clear) dose-related response. Moreover, values in
treated animals were within normal limits. Therefore, these findings were considered to be unrelated to treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related alterations in organ weights. The statistically significant change noted in relative spleen weights for females at 300 mg/kg was unrelated to treatment because this occurred in the absence of a dose-related response and no corroborative findings were observed.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related gross observations. All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this strain and age, and did not show a dose-related incidence trend. These findings were therefore considered to be unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Microscopic change was noted in the heart of 3/5 males at 1000 mg/kg, consisting of myofiber necrosis (in the apex and/or left papillary muscle) up to slight degree.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
Dose response relationship:
Relevant for humans:
not specified

Any other information on results incl. tables

 Dose level (mg/kg)  0  100  300  1000
  Heart (number examined)  5  5  5  5
Necrosis, myofiber        
 Minimal  -  -  -  2
 Slight  -  -  -  1

Applicant's summary and conclusion

The following parental NOAEL of 300 mg/kg was identified based on a degenerative change in the heart (myofiber necrosis in the apex and/or left papillary muscle) of males treated at 1000 mg/kg.
Executive summary:

In a subacute toxicity study the substance was administered to 10 rats/sex/dose in by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. There were no substance-related effects in mortality, clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, or gross pathology. Histopathological examination revealed a degenerative change in the heart (myofiber necrosis in the apex and/or left papillary muscle) of males treated at 1000 mg/kg. The NOAEL is  300 mg/kg bw/day based on the degenerative change in the heart at the top dose in males. This subacute toxicity study in the rat is acceptable and satisfies the guideline requirement for a subacute oral study OECD 422 in rats.