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Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 July 2017 - 21 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes (incl. certificate)
Limit test:
no
Justification for study design:
Species: Recognized by international guidelines as a recommended test system.
Dose levels: It was considered a suitable dose level range, based on the preliminary results obtained in the previous non-GLP study PQ36XP 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at in Envigo CRS, S.A.U.:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 021340
- Expiration date of the lot/batch: July 2022
- Purity test date: 25 August 2003
- Appearance: Red powder


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at ambient conditions in the dark (away from direct light).
- Stability under test conditions: 8 days
- Solubility and stability of the test substance in the solvent/vehicle: Yes
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Recognized by international guidelines as a recommended test system. Also selected for consistency with species used in previous toxicological studies.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation:
Males: 10 weeks
Females: 10 weeks

- Weight at study initiation:
Males: 256-359 g
Females: 186-248 g

- Housing:
Cages with standard, granulated, S8-15 sawdust bedding.
Premating period (four animals/cage) Makrolon type-IV cages
Mating period (one male and one female/cage) Makrolon type-III cages
Postmating, gestation and lactation periods (individual) Makrolon type-III

- Diet: ad libitum, standard commercial diet
- Water: ad libitum, tap water
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:


ENVIRONMENTAL CONDITIONS
- Temperature (°C):
20 - 24
- Humidity (%):
between 30 and 70
- Air changes (per hr):
15-20
- Photoperiod (hrs dark / hrs light):
12 fluorescent light/12 hours dark
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on exposure:
The necessary amount of test item was weighed in a disposable flask. 70-80% of vehicle (Arachis oil) required to reach the final formulation volume was poured into an empty disposable flask and the test item was added. The content of the flask was mixed using an Ultra-Turrax or a homogenizer (depending on the total volume of formulation prepared) until complete homogenization. The suspension was then transferred to a volumetric vessel. A small amount of vehicle was used to remove any formulation remaining in the disposable flask and the mixture was poured into the vessel. Vehicle was then added to the volumetric vessel to make up to volume. Finally, the suspension was transferred to an appropriate container and submitted to magnetic stirring for 10 minutes before sampling for dilution. Dilution Preparation: Intermediate and Low concentration Formulations (Groups 2 and 3) It was prepared by diluting the high formulation and submitted to magnetic stirring for 10 minutes before aliquoting.
The density was taken the first day of preparation of formulation. Formulations were maintained under agitation for 3 ¾ to 24 hours until the end of the administration, assuring that formulations were homogenous during their administration.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 1-4 days
- Proof of pregnancy:Ejected copulation plugs. Sperm within vaginal smear referred to as [day 0 / day 1] of pregnancy
Day 0 of gestation: When positive evidence of mating detected.
Male/female separation: day when mating evidence detected.
Pre-coital interval: Calculated for each female as time between first pairing and evidence of mating.
Females showing no evidence of pregnancy (female 95 at 300 mg/kg/day and females 105 and 107 at 1000 mg/kg/day) were sacrificed 26 days after the last day of the mating period.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The formulations prepared at three different concentrations were analyzed twice over the course of the study to verify correct preparation.
Control (vehicle) formulations were analyzed to confirm the absence of test item or other substances at the retention time of the test item.
The test item was used as analytical standard.
Results were expected to be within ±20% of nominal value and the coefficient of variation (CV) should be ≤10%.
Duration of treatment / exposure:
5-8 weeks
Frequency of treatment:
daily
Details on study schedule:
- F1 parental animals not mated until 2 weeks after selected from the F1 litters.

- Age at mating of the mated animals in the study: 12 weeks minimum
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
control
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:
12
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: It was considered a suitable dose level range, based on the preliminary results obtained in the previous non-GLP study PQ36XP 14-day Oral (Gavage) Dose-Range Toxicity Study for OECD 422 conducted at in Envigo CRS, S.A.U.:
- The high dose was selected as no toxicity was observed in the preliminary study at 1000 mg/kg/day and considering it as a limit dose to be tested.
- Intermediate and low dose levels were selected considering approximately a 3-fold interval between doses.
- Rationale for animal assignment (if not random): random
- Other: The animals were allocated at random to four treatment groups. Spare animals were used and/or animals were exchanged. The rejected animals took no further part in the study after commencement of treatment.
Positive control:
None
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: day 1, and weekly thereafter

OTHER:Sensory Reactivity and Grip Strength, Motor Activity,
Oestrous cyclicity (parental animals):
Using a inoculation loops during the following phases:
• For 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study.
• Daily from the beginning of treatment period until evidence of mating.
• On the day of necropsy
A cotton swab impregnated in physiological saline was used in order to check the evidence of mating during the mating period.
Sperm parameters (parental animals):
not specified
Litter observations:
Clinical observations, litter size, sex ratio, Individual offspring body weights, ano-genital distance, nipple/areolae count
Postmortem examinations (parental animals):
Organ Weights, Macroscopic Pathology
Postmortem examinations (offspring):
Premature decedents,
Statistics:
All statistical analyses were carried out separately for males and females. The following comparisons were performed: Group 1 vs. 2, 3 and 4.
For continuous data, a parametric analysis was performed if Bartlett's test for variance homogeneity is not significant at the 1% level. Treated groups were compared to control using Williams' test, unless there is evidence against a monotonic dose-response when Dunnett's test was performed instead.
A non-parametric analysis was performed if Bartlett's test is still significant at the 1% level following both logarithmic and square-root transformations. Treated groups were compared to control using Shirley's test, unless there is evidence against a monotonic dose response when Steel's test was performed instead.
Reproductive indices:
Percentage mating, Conception rate, Fertility index
Offspring viability indices:
Post-implantation survival index, live birth index, viability index
Clinical signs:
no effects observed
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):
not examined
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
Description (incidence and severity):
Statistically significant differences from controls (hemoglobin at 1000 mg/kg/day in males and females, mean cell hemoglobin at 1000 mg/kg/day in females, mean corpuscular volume at 100 and 1000 mg/kg/day in females and at the doses of 300 and 1000 mg/kg/day in large unstained cells in females and in monocytes in males) were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Blood chemistry after 6-7 weeks of treatment in males and on day 16 of lactation revealed statistically higher than control creatinine levels at 300 and 1000 mg/kg/day in males and lower than control bile acids in males at 1000 mg/kg/day. A trend to higher urea mean values in the test item administered groups was observed in males and females. Females receiving 1000 mg/kg/day had statistically higher urea values than controls. Statistically significant higher potassium, chloride and calcium means were recorded in males at 1000 mg/kg/day when compared to control. The higher chloride mean value was also significant compared to control at 300 and 1000 mg/kg/day in males. As a general rule, increased albumin and albumin/globulin ratio mean values at 300 and 1000 mg/kg/day were recorded in both sexes (they were statistically significant at1000 mg/kg/day for albumin and for albumin/globulin ratio at 300 and 1000 mg/kg/day).
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Sensory reactivity conducted during week 5 in males and between days 7-9 of lactation in females revealed no findings which were considered treatment related.
Grip strength recordings revealed a significant dose-related increase in mean hind limb values in females. However there was no evidence observed in clinical signs that can corroborate this finding and it was not observed in males.
The motor activity assessment conducted during week 5 in males and between days 7-9 of lactation in females revealed no treatment related effects in males and females at all dose levels.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Absolute and body-weight-adjusted adrenal weights in males and kidney weights in females were higher than control in all test item administered groups (100, 300 and 1000 mg/kg/day) and attained significant differences when compared to control. Differences were dose related. Statistically significant differences were observed at 300 mg/kg/day and 1000 mg/kg/day in ovaries.
Gross pathological findings:
no effects observed
Description (incidence and severity):
The macroscopic examination performed at the end of the treatment period revealed no test item- related changes in males or females. The internal pinkish coloration observed in all test item administered groups is related with the test item color.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes that were considered related to treatment with the test item were seen in:
- Kidneys (renal cortex): minimal to moderate tubular basophilia in both genders in all test item administered groups. Tubules of the most affected males showed karyomegaly and were accompanied by minimal, multifocal, interstitial mononuclear
cell infiltrates.
- Thymus of both sexes at 1000 mg/kg/day: minimal involution/atrophy or minimally increased cortical apoptosis was seen and cortical hypertrophy was also observed in the affected females.
- Adrenal glands of females at 1000 mg/kg/day: bilateral, minimal to slight diffuse cortical hypertrophy.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
All females allocated to the study showed regular 4 day estrus cycle prior to the start of treatment and during treatment
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Description (incidence and severity):
At termination, all reproductive phase females (pregnant) showed diestrus with the exception of 2/12 at 100 mg/kg/day, 1/11 at 300 mg/kg/day and 2/9 at 1000 mg/kg/day, which showed different stages of estrous cycle, indicating that these females had recovered estrous cycle again. There was no effect of treatment on the pre-coital interval: all animals mated within four days of mating at the first estrous.
The changes observed in organ weights in ovaries when compared to control were considered to be incidental taking into account the magnitude of change, that there was no dose relationship and that there was no correlation with clinical pathology or histopathology.
Estrous cycles and reproductive parameters of pre-coital interval, mating performance, fertility or gestation length or index were unaffected by treatment.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food efficiency
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive function (oestrous cycle)
reproductive performance
Key result
Critical effects observed:
no
System:
other: kidney
Organ:
other: various
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:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no macroscopic findings observed in the offspring that died prior to the scheduled termination or among those offspring killed on days 4 or 13-15 of age that were attributable to maternal treatment with Lumière Pink S.M. 8135N.
Histopathological findings:
no effects observed
Other effects:
not examined
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
There was no effect of treatment on litter size or sex ratio.
There were no clinical signs observed among the F1 litters that could be clearly attributable to parental treatment with Lumière Pink S.M. 8135N.
There was no effect of treatment on mean body weights of male and female offspring on day 1 of age or on subsequent body weight measurements until day 13 of age.
Mean ano-genital distance in male offspring was similar in the test item dosed groups and slightly higher than in controls. Despite the fact that the differences recorded were statistically significant they were not considered dose related, thus these changes were devoid of any toxicological relevance. There was no effect recorded in male nipple observations.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
clinical signs
mortality
body weight and weight gain
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no
System:
other: various
Organ:
other: various
Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Conclusions:
In conclusion, the effects of oral (gavage) administration of Lumière Pink S.M. 8135N to Wistar rats receiving 100, 300 or 1000 mg/kg/day for 14 days prior to mating and until sacrifice can be summarized as follows:

Reproductive / developmental toxicity:
- The No Observed Adverse Effect Level (NOAEL) for reproductive / developmental toxicity was considered to be 1000 mg/kg/day, taking into account that there was no effect on estrous cycle, pre-coital interval, mating performance, fertility and gestation
length or in the offspring on litter size, survival, sex ratio, clinical signs, body weights, ano-genital distances or macropathology.
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:
Guideline study Klimisch 1
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test substance Lumière Pink S.M. 8135N is not classified for toxicity to reproduction.