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Effects on fertility

Description of key information

A repeated dose/reproductive screening study on rats conducted in accordance with the OECD 422 guideline and GLP provides suitable information on possible effects of the test substance on fertility.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
This strain is widely used in toxicity studies using rodents, there is abundant historical data and a large number of animals are available.
Route of administration:
oral: gavage
Vehicle:
other: Methylcellulose
Remarks:
0.5 w/v% in water for injection
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Dosing solutions were prepared once every 9 days as observed to be stable over this period. These were stored refridgerated ahead of use each day. For the high dose, a set amount of the substance was weighed and ground using a pestle and mortar. The vehicle was gradually added and the suspension added to a measuring cylinder. All equipment was washed with vehicle and added to the cylinder. This was brought upto a final volume in order to prepare a concentration of 0.2 mg/mL. The dosing preparations were well mixed. For the intermediate and low dose, a set amount of the high dose formulation was added to measuring cylinders and made up with vehicle in order to prepare 0.05 and 0.0125 mg/mL formulations. These were also well mixed. All dosing formulations were appropriately identified.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Selected based on work conducted in preliminary repeated dose study and a seperate homogeniety and stability test. Methyl cellulose is a standard vehicle for tests of this type.
- Concentration in vehicle: 0.0125, 0.05 and 0.2 mg/mL
- Amount of vehicle (if gavage): 10 mL/Kg
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged (how): Individually in polymethylpentene cages
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At the initial preparation analytical samples were taken from the low, mid and upper layer of the whole dosing formulation at each concentration. These were then analysed by HPLC to ensure the dose formulations were homogeneous
Duration of treatment / exposure:
Males: 42 days in total
Females: 14 days before mating, until Day 13 of lactation.
Recovery females: 42 days in total
Frequency of treatment:
Once daily between 8:01 and 12:36.
Details on study schedule:
- Age at mating of the mated animals in the study: approximately 11 weeks (parental). No mating of the F1 generation occured as is not a requirement of the testing guideline.
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
0.125 mg/kg bw/day (nominal)
Dose / conc.:
0.5 mg/kg bw/day (nominal)
Dose / conc.:
2 mg/kg bw/day (nominal)
No. of animals per sex per dose:
As per Table in 'Any other information on materials and methods incl. tables' section.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected based on the results of a 14 day preliminary study. Doses of 30 mg/kg were deemed to severe to rats with death and changes following corrosion noted. Changes at 6 mg/kg were less severe but changes noted in the stomach's of animals were similar to that observed at 30 mg/kg.
- Rationale for animal assignment (if not random): Vaginal smears were collected from females 9 days ahead of the start of dosing and the oestrous cycles observed. Some animals presented with abnormalities and were not selected for use on study. All other animals were randomly allocated to study based on bodyweight (within 20% of the sex mean).
- Rationale for selecting satellite groups: Satellite animals were included on study in the control and high dose groups. These were included to assess the potential for recovery of effects that may have been observed on study.
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
Not required
Parental animals: Observations and examinations:
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Oestrous cyclicity (parental animals):
For females, vaginal smears were collected with a swab for 9 days from the end of quarantine period, and the oestrous cycle was observed. This information was used to assist in the selection of suitable females for use on study.

Vaginal smears were also collected with swabs from all test females in the morning (same time every day) from the initial dosing day to the day of successful copulation or end of the mating period to confirm the estrous cycle. The obtained smears were collected on a plate for each animal, and stained with Giemsa. The estrous cycle was classified into diestrus (D), proestrus (P), estrus (E) and metestrus (M).

The mean estrous cycle (number of days from the estrous period to the next estrous period) and the number of the estrous period during the test period were calculated.
Sperm parameters (parental animals):
Parameters examined in male parental generation:

Testis weight and epididymis weight were measured in parental males. No further assessment of sperm parameters were taken.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: On PND 4, the litter size was standardized to 8 (4/sex/litter) by random removal of offspring. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 regardless of the sex ratio. Litter with less than 8 offspring was maintained as is.

The offspring culled at the litter size adjustment (offspring excluded from blood collection) were euthanized by overdose with pentobarbital sodium (undiluted solution, 0.05 to 0.1 mL/body) via an intraperitoneal injection and discarded. Offspring not culled after standardization were
allocated for the collection of blood samples.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
Anogenital distance, nipple development, number of litter (numbers of live newborn and stillborn), sex, the presence of external abnormalities, mortality and bodyweight.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external abnormalities; possible cause of death was not determined for pups born or found dead.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: Not conducted in pups (not a guideline requirement)

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: Not conducted in pups (not a guideline requirement)
Postmortem examinations (parental animals):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed on postnatal day 13.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of external examinations including the cervical, thoracic, and abdominal viscera. The offspring subjected to blood sampling were necropsied after blood sampling by decapitation after anesthetizing by inhalation of isoflurane. Other animals were euthanized by exsanguination from the lateral iliac artery under anesthesia with pentobarbital sodium via the intraperitoneal injection and necropsied. Furthermore, the thyroid glands of 1 male and 1 female (animals subjected to blood sampling for hormone level measurement) per litter were collected and fixed and preserved in 10 vol% phosphate buffered formalin.

Statistics:
For the following numeral data, mean values and standard deviations were calculated in each group. Bartlett’s test was performed to compare variances among groups (significance level: 5%). When variance of data was homogeneous, Dunnett’s multiple comparison test was performed to compare with the control group. When variance of data was heterogeneous, Steel’s multiple comparison test was performed to compare with the control group.

Oestrous cycle, number of oestrus, days until copulation, gestation length, number of implantations, number of delivered offspring, number of live offspring, body weight of offspring and AGD.

The following items were tested with the chi-square test for comparison between the control and each test substance group.

Copulation index, fertility index, gestation index, delivery index and sex ratio.

The following items were tested with the Wilcoxon’s rank sum test for comparison between the control and each test substance group.

Stillborn index, external anomaly index, external anomaly typing index, birth index, viability index on Days 4 and 13 and nipple development anomaly index.
Reproductive indices:
Days until copulation, copulation index (%), fertility index (%), gestation length, gestation index (%) and delivery index (%).
Offspring viability indices:
Birth index (%), stillborn index (%), viability index on Days 4 and 13 (%), sex ratio, external anomaly index (%), external anomaly typing index (%) and nipple development anomaly index (%).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Details included in the Endpoint study record for the repeated dose aspect of this study (cross referenced).
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in the mean estrous cycle or count of oestrus, indicating no effect of the test substance on prolongation or shortening of the oestrous cycle.

Besides, irregular oestrous cycle was noted in 1 female of the 2 mg/kg group. However, this was judged not to be treatment-related as the copulation and conception were confirmed. Irregular oestrous cycle was also noted in 1 female of the control group; in which abnormalities were noted in the ovary and uterus
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No effects were noted on the organ weights of the testis or epididymides. No effects were noted pathologically.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
No significant difference was noted in the copulation index or fertility index between the control and test substance groups.

Besides, shortening of duration of copulation was noted in the 2 mg/kg group. However, this was judge not to be treatment-related as the mating was confirmed in 7/12 animals at the 1st oestrus in the 2 mg/kg group. No mating was confirmed in 1 female in the 2 mg/kg group. At necropsy, no abnormality was noted in the genital organs of this animal and the male counterpart. Although the cause of non-copulation was not clear, the change was judged not to be treatment-related for its incidence. Non-copulated female was also noted in 1 female of the control group. The cause of non-copulation was abnormal findings in the ovary and uterus. Non-pregnancy was noted in 1 female of the 2 mg/kg group. In the male counterpart, abnormalities were observed in the testis and epididymis; indicating spontaneous changes. Therefore, non-pregnancy was judged not to be treatment-related. Non-pregnancy was also noted in 1 female of the 0.125 mg/kg group. However, this was judged not to be treatment-related as the state of incidence.

No treatment-related change was noted in the delivery or nursing. Total litter loss was observed in 1 dam of the 0.5 mg/kg group on LD 3. This was judged not to be treatment-related.

No significant difference was noted in the gestation length, number of implantations and litter, delivery index or gestation index.
Overall, there were no treatment related effects on paternal animals when considering reproductive performance.
Dose descriptor:
NOAEL
Effect level:
2 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects noted and accordingly NOAEL is set as the top dose.
Critical effects observed:
no
Clinical signs:
no effects observed
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
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:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in anogenital distance or nipple development.

Low values of relative AGD value were noted in male offspring of the 2 mg/kg group. However, this was judged not to be treatment-related as the change was within mean ± 2 S.D. of the historical data at the test facility and no related change was noted in the plasma total T4 concentration.
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related change was noted in any offspring.

Visceral inversion was observed in 1 male of the 0.125 mg/kg group, indicating spontaneous change.
Histopathological findings:
not examined
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
No significant difference was noted in the plasma total T4 concentration between the control and test substance groups.
Overall no treatment-related effects were noted in the offspring of parental animals treated with the test substance.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
2 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No toxicologically relevant effects noted and accordingly NOAEL is set as the top dose.
Critical effects observed:
no
Reproductive effects observed:
no
Conclusions:
A reliable OECD 422 study is available providing data on fertility of parental animals and offspring development. This study showed no effects at the highest feasible dose (due to the corrosive nature of the substance) on parental animal reproductive performance or offspring indices and accordingly the high dose is set as the NOAEL.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A reliable OECD 422 study conducted to GLP and OECD guidance is available.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

No toxicologically significant effects were observed on study and the NOAEL was set as 2 mg/kg (the maximum dose tested) when considering parameters relative to reproduction.

Effects on developmental toxicity

Description of key information

A repeated dose/reproductive screening study on rats conducted in accordance with the OECD 422 guideline and GLP provides suitable information on possible effects of the test substance on development.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
2 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
A reliable OECD 422 study conducted to GLP and OECD guidance is available.
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

No toxicologically significant effects were observed on study and the NOAEL was set as 2 mg/kg (the maximum dose tested) when considering parameters relative to development.

Justification for classification or non-classification

A reliable OECD 422 study is available which provides a NOAEL of 2 mg/kg (the highest feasible dose tested as a result of the corrosive properties of the substance). No treatment related effects on reproduction or development were noted on study and accordingly classification is not warranted for this substance based on the available data in accordance with the CLP regulation (EC No. 1272/2008, as amended).

Additional information