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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant, guideline study, available as an unpublished study report. Acceptable for assessment.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Solid
Details on test material:
- Name: Fatty acids C18-(unsaturated) lithium salts
- Substance type: Technical product
- Physical state: Solid
- Lot/batch No.: SF_173 (#163879)
- Expiration date of the lot/batch: 21 January 2012
- Storage condition of test material: Room temperature, protected from light

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: Males - 266-311g; Females (nulliparous and nonpregnant) - 191-222g
- Housing: Individually housed in suspended, stainless steel, wire-mesh type cages, except during pairing, near parturition and during lactation. P females moved to plastic caging with wood chip bedding from GD20 - LD 4. Animal enrichment per SOP-ACU-89
- Diet (e.g. ad libitum): Lab Diet Certified Rodent Diet #5002, PMI Nutrition International, Inc., ad libitum.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 64-79
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 h /12 h

IN-LIFE DATES: From: 30 November 2010 to 1 Febraury 2011

Administration / exposure

Route of administration:
dermal
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Dorsal surface; equivalent to approx. 10% of the total body surface area
- Type of wrap if used: Gauze dressing and non-absorbent cotton covered with self-adhesive tape and athletic tape.
- Time intervals for shavings or clipplings: As required

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Wypall wet with distilled water.
- Time after start of exposure: 6 h

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.5 mL/kg
- Concentration (if solution): equivalent to 0; 100; 300; 1000 mg/kg/day (nominal)
- Constant volume or concentration used: Yes


VEHICLE
- Distilled deionised tap water
- Concentration (if solution): N/A
- Lot/batch no. (if required): Not available
- Purity: Not available

USE OF RESTRAINERS FOR PREVENTING INGESTION: No
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of fatty acids C18-(unsaturated) lithium salts in 52 dosing formulation samples was established by determining lithium content by Inductively Couple Plasma Optical Emission Spectroscopy (ICP-OES). Samples were processed by microwave aided acid digestion (DIN 51460-2) before submitting to ICP-OES (EN ISO 11885).
Details on mating procedure:
After 2 weeks of treatment, P animals (Groups 1 to 4), each female was housed in cage of a male from the same treatment group.
- M/F ratio per cage: 1
- Length of cohabitation: Until evidence of copulation was established (max 14 days). Housed individually during daytime 6-hour exposure period.
- Proof of pregnancy: vaginal plug and / or vaginal lavage with the presence of sperm; referred to as gestation day (GD) 0
- After 14 days of unsuccessful pairing females were returned to individual cages.
- After successful mating each pregnant female was caged individually for the remainder of the study.
Duration of treatment / exposure:
Males dosed for at least 43 days, beginning 14 days prior to mating (Groups 1-4).
Dosing of the females began 14 days prior to mating and continued through mating, up to and including GD 19 (Groups 1-4). Another set of animals (Groups 5 and 6) were dosed for a total of 43 days and then began a 14-day (non-treated) recovery period.
Frequency of treatment:
Daily, once per day for 6 hours.
Duration of test:
Terminal Groups (Groups 1 to 4): Dosed for at least 43 days
Recovery Groups (Groups 5 and 6): Dosed for a total of 43 days before beginning a 14-day (non-treated) recovery period
No. of animals per sex per dose:
Terminal Groups (Groups 1 to 4): 10 animals per sex per dose
Recovery Groups (Groups 5 and 6): 5 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected on the basis of available data from a previous dose ranging study (MPI Study No. 1883-001). Animals were dosed dermally once a day at 0, 525, 1050 and 2100 mg/kg bw/day (water based Fatty acids C18-(unsaturated) lithium salts) and for two weeks at 2106 mg/kg bw/day with oil-based Fatty acids C18-(unsaturated) lithium salts. The results of this study identified significant dose responsive dermal effects, based on maximized erythema and eschar scores at dose levels ≥1050 mg/kg bw/day fatty acids C18-(unsaturated) lithium salts. A slight decrease in mean body weight was noted in males during the last week of dosing, however, not other adverse body weight or food consumption effects were observed at the dose levels tested. A nominal high-dose level of 1000 mg/kg bw/day (at a slightly lower concentration based on dose volume) was selected for the defintive study to insure some morbidity. The nominal low dose level (100 mg/kg bw/day) was selected with 10-fold decrease to insure a no effect level, and a nominal mid-dose level of 300 mg/kg bw/day was selected to be about 3 times higher than the low-dose level and about 3 times lower than the high-dose level.
- Rationale for animal assignment (if not random): All animals placed on study will have body weights that fall within 20% of the mean bodyweight for each sex. Animals considered suitable for study will be weighed prior to treatment. After the appropriate number of animals with the highest and lowest body weights has been excluded, the remaining required number of animals on study will be randomized, by sex, into treatment groups using a standard, by weight, measured value randomization procedure
- Rationale for selecting satellite groups: As above
- Post-exposure recovery period in satellite groups: 14-day non treatment period
- Section schedule rationale (if not random): Randomized

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice a day, 7 days a week, for morbidity, mortality, injury, and availability of food and water. Towards end of gestation period, P females examined twice daily for signs of parturition.
- Cage side observations recorded are not reported, but are maintained in the study file.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the study (after wrap removal on dose days).

DERMAL IRRITATION: Yes
- Time schedule for examinations: Prior to first dose and daily during the study (after wrap removal on dosing days).

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly during the study. Mated females weighed on GD 0, 7, 14 and 20 and on LD 0 and 4 and at termination.

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

WATER CONSUMPTION: No

OTHER:
HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination / at recovery
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: No
- How many animals: 5 per sex per group (P animals) in Groups 1-4 at Termination (same animals designated for behavioral testing). 5 per sex per group in Groups 5 and 6 at Recovery
- Parameters checked: Leukocyte count (total and absolute differential); erythrocyte count; hemoglobin; hematocrit; mean corpuscular hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin concentration (calculated); absolute reticulocytes; platelet count; blood cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination / at recovery
- Animals fasted: No
- Number of animals: 5 per sex per group (P animals) in Groups 1-4 at Termination (same animals designated for behavioral testing). 5 per sex per group in Groups 5 and 6 at Recovery
- Parameters checked: Alkaline phosphatase; total bilirubin (with direct bilirubin if total bilirubin exceeds 1 mg/dL); aspartate aminotransferase; alanine aminotransferase; gamma glutamyl transferase; sorbitol dehydrogenase; urea nitrogen; creatinine; total protein; albumin; globulin and A/G (albumin/globulin) ratio (calculated); glucose; total cholesterol; triglycerides; electrolytes (sodium, potassium, chloride); calcium; phosphorus
Ovaries and uterine content:
No data reported
Fetal examinations:
Litters were examined as soon as possible after delivery(LD 0) and on LD 4. The F1 offspring sacrificed on LD 4 and these animals were subjected to postmortem external examinations.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, number of stillborn and liveborn pups, survival, number of males and females, individual body weights, abnormal behaviour, and gross abnormalities of the pups.

GROSS EXAMINATION OF DEAD PUPS: Yes, for external abnormalities. Offspring found dead on LD 0 had lungs removed and placed in tap water to determine whether live/stillborn.
Statistics:
The raw data were tabulated within each time interval, and the mean and standard deviation were calculated for each endpoint by sex and group. For each endpoint, treatment groups were compared to the control group using: Group pair-wise comparisons; Cochran Manzel Haenszel test; log transformation / group pair-wise comparisons; Fisher's exact test; Arcsin-square root transformation, and; Covariate analysis.
Indices:
Live Birth Index (%) = (No. live pups at birth / No. pups born) x 100
Stillborn Index = No. stillborn pups / No. pups born
Viability Index – Day 4 (%) = (No. pups surviving 4 days (preculling) / No. live pups at birth) x 100
Historical control data:
No data reported

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects: No effects
Reproductive parameters (male/female fertility and fecundity indices, and copulatory intervals) were unaffected by treatment. No statistically significant changes in delivery data and pup development / survival.

Details on maternal toxic effects:
- Clinical signs (parental animals): 345 and 1089.75 mg/kg bw/day: localized scabbed area on dorsal surface and scabbed area in the thoracic region (related to test site) during dosing period. Recovery animals indicated trend towards recovery after cessation of dosing.
- Body weight and food consumption (parental animals): Trend to decreasing mean body weight in males at 1089.75 mg/kg bw/day - though not statistically significant. Mean body weight change was statistically decreased in treated males at 1089.75 mg/kg bw/day during the premating interval from Weeks 1 to 3.
- Test substance intake (parental animals): Not examined
- Reproductive function: Estrous cycle (parental animals): Not examined
- Reproductive function: Sperm measures (parental animals): Not examined
- Reproductive performance (parental animals): Male/female fertility and fecundity indices and copulatory intervals unaffected by treatment.
- Organ weights (parental animals): Relative mean spleen weight increased in 1089.75 mg/kg/d males. Considered to be secondary adaptive response to localised dermal effects
- Gross pathology (parental animals): Test article-related macroscopic findings (terminal necropsy): minimal to moderate abrasion/scab formation in control males and treated animals of both sexes. Dose-related increase in incidence/severity in females (all doses) and males (345-1089 mg/kg/d)
- Histopathology (parental animals): Test article-related microscopic findings in skin: minimal/moderate erosion/ulceration, epidermal hyperplasia and exudate, minimal/mild acute/subacute/chronic inflammation, minimal edema. 1000 mg/kg/day: secondary adaptive findings in thymus and spleen.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Remarks:
Generation P male/female
Effect level:
> 1 089.75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
Generation P male/female
Effect level:
111.25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: NOAEL for local effects at test site based on observations: minimal to moderate erosion/ulceration, epidermal hyperplasia and exudate; minimal to mild acute to subacute/chronic inflammation; and minimal edema at 300 and 1000 mg/kg bw/day dose groups.
Remarks on result:
other: The 1000 mg/kg bw/day recovery group animals indicated partial resolution of these findings following the 14 day recovery period.

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No reproductive effects or definitive test-article related changes in the development or survival of the offspring.

- Viability (offspring): No effects
- Clinical signs (offspring): No effects
- Body weight (offspring): No effects
- Sexual maturation (offspring): Not examined
- Organ weights (offspring): Not examined
- Gross pathology (offspring): No effects
- Histopathology (offspring): Not examined
- Details on results (offspring): There were no treatment-related effects at any dose level on any of the reproductive parameters evaluated in this study including offspring survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and pup sex ratio. There were also no treatment-related effects on any of the developmental parameters evaluated including external abnormalities, number of live and still births, mortality, sex determination and weights of pups.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
> 1 089.75 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No reproductive effects or definitive test-article related changes in the development or survival of the offspring.

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Dermal application of fatty acids C18-(unsaturated) lithium salts to the dorsal surface of Parental male and female rats at dose levels of 100, 300, and 1000 mg/kg bw/day did not result in any adverse reproductive effects or any definitive test article-related changes in the development or survival of the offspring.

In addition, results from the clinical pathology evaluations and neurobehavioral testing did not reveal any definitive effects that could be attributed to treatment at the dose levels tested. Test article-related dermal changes were limited to local effects at the test site for Parental animals at 300 and 1000 mg/kg bw/day, which showed a decreasing trend following cessation of dosing in the designated recovery animals. Microscopic evaluation of the skin at the test site showed minimal to moderate erosion/ulceration, epidermal hyperplasia and exudate, minimal to mild acute to subacute/chronic inflammation and minimal edema, which confirmed the macroscopic findings. At recovery necropsy, test article-related microscopic findings in treated skin in animals at 1000 mg/kg bw/day were morphologically similar, although they occurred at a lower incidence and severity indicating partial resolution of these findings following the recovery period. Microscopic findings of splenic extramedullary hematopoiesis and thymic lympoid depletion noted at 1000 mg/kg bw/day were considered to be secondary adaptive responses to parturition and localized dermal effects.

Systemic effects were limited to changes in body weight and organ weight at 1000 mg/kg bw/day, the highest dose level tested.

In summary, there were no treatment related effects at any dose level on any of the reproductive parameters evaluated in this study, or in any of the developmental parameters evaluated. Based on these daya, the NOAEL for developmental toxicity was >1089.75 mg/kg bw/day and the NOAEL for reproductive toxicity was also >1089.75 mg/kg bw/day.

Applicant's summary and conclusion

Conclusions:
Dermal application of fatty acids C18-(unsaturated) lithium salts to the dorsal surface of Parental male and female rats at dose levels of 100, 300, and 1000 mg/kg bw/day did not result in any adverse reproductive effects or any definitive test article-related changes in the development or survival of the offspring.
Executive summary:

The developmental toxicity of fatty acids C18 (unsaturated) lithium salts was assessed in a combined repeated dose and reproductive toxicity screening test following OECD guideline 422 (MPI 2011). The parental generation was dermally administered daily for 6 hours per day with fatty acids C18 (unsaturated) lithium salts at concentrations of 100, 300 and 1000 mg/kg bw/day. The offspring of the treated rats were then assessed for survival (gestation and postnatal survival indices, percent pre- and post-implantation loss), pup body weight and sex ratio and external abnormalities.