Calcium didocosanoate

Developmental toxicity / teratogenicity

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other: GLP-compliant, guideline study, available as an unpublished study report.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

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 422; GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system 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

Type of coverage:

semiocclusive

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 take 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).

Duration of treatment / exposure:

Males dosed for at least 43 days, beginning 14 days prior to mating (Groups 1-4).
Dosing of the females will begin 14 days prior to mating and continue through mating, up to and including GD 19 (Groups 1-4). Another set of animals (Groups 5 and 6) will be dosed for a total of 43 days and will then begin a 14-day (non-treated) recovery period.

Frequency of treatment:

Daily, once per day for 6 hours.

Remarks:

Doses / Concentrations:
0; 100; 300; 1000 mg/kg bw
Basis:
nominal per unit body weight

Remarks:

Doses / Concentrations:
0; 111.25; 345; 1089.75 mg/kg bw
Basis:
analytical per unit body weight

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 by the Sponsor 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/day (water based Fatty acids C18-(unsaturated) lithium salts) and for two weeks at 2106 mg/kg/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/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/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/day) was selected with 10-fold decrease to insure a no effect level, and a nominal mid-dose level of 300 mg/kg/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.

Positive control:

Not included

Observations and examinations performed and frequency:

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.
- 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 dosing days).

DERMAL IRRITATION (if dermal study): 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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

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
- 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:
 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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once pretest, weekly and on GD 0, 7 and 20 (after wrap removal on doing days).
- Dose groups that were examined: all animals in all dose groups
- Observations included, but were not limited to, changes in the skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions, and autonomic activity (i.e., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Changes in gait, posture and reactivity to handling, as well as the presence of clonic or tonic movements, sterotypics (i.e., excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behavior (i.e., self-mutilation, walking backwards) were also recorded.

FUNCTIONAL OBSERVATIONAL BATTERY OBSERVATIONS
- Time schedule for examinations: During last week of treatment for the males amd on LD3 in females (only lactating females evaluated).
- Dose groups that were examined: dose groups 1-4 only, 5 per dose group per sex.
- Battery of functions tested: (1) autonomic function (Ranking of the degree of lacrimation and salivation, with a range of severity scores from none to severe. Presence or absence of piloerection and exophthalmus. Measurement of urination and defecation including polyuria and diarrhea. Pupillary function as indicated by constriction of the pupil in response to light.); (2) Description, incidence and severity of convulsions, tremors, or degree of palpebral closure, e.g., ptosis, abnormal motor movements, both in the home cage and the open field; (3) Ranking of the subject's reactivity to general stimuli such as removal from the cage or handling, with a range of severity from no reaction to hyperactivity; (4) Ranking of the subject's arousal level during observations of the unperturbed subject in the open field, with a range of severity scores from coma to hyperalertness; (5) Descriptions and incidence of posture abnormalities observed in the home cage and incidence of gait abnormalities observed in the open field; (6) Ranking of any gait abnormalities, with a range of severity scores from none to severe; (7) Forelimb and hindlimb grip strength measured using the procedure described by Meyer; (8) Quantitative measure of landing foot (hindfoot) splay as described by Edwards and Parker; (9) Sensorimotor responses to stimuli of different modalities will be used to detect gross sensory deficits. Pain perception will be assessed by ranking the reaction to a tail pinch and measuring the latency to a nociceptive (thermal) response when the subject is placed on a heated (52+/- 1 degree C) surface, as described by Ankier. The response to a mechanically produced "click" will be ranked to assess audition and reactivity; (10) Body weight; (11) Description and incidence of any unusual or abnormal behaviors, excessive or repetitive actions (stereotypies), emaciation, dehydration, hypotonia or hypertonia, altered fur appearance, red or crusty deposits around the eyes, nose, or mouth, and any other observations that may facilitate interpretation of the data; (12) Other measures to be recorded are count of rearing activity in the open field, ranking of air righting, body temperature measured rectally, spontaneous or excessive vocalizations, alterations in rate and ease of respiration (e.g. rales or dyspnea), and sensorimotor responses to visual (approaching blunt object) and proprioceptive (touch on rump with blunt object) stimuli.

MEASUREMENT OF ACTIVITY AND EMOTIONALITY
Time schedule for examinations: During last week of treatment for the males amd on LD3 in females (only lactating females evaluated).
- Dose groups that were examined: all dose groups, 5 per dose group per sex.
- The following activity was recorded:
 Horizontal and vertical activity counts
 Total horizontal distance traveled
 Number of runs in a clockwise pattern
 Number of runs in a counterclockwise pattern
 Rest time
 Time spent in nonambulatory movements such as grooming
 Time spent in whole-body ambulatory movements
 Number of separate nonambulatory movements conducted
 Number of separate horizontal movements conducted
 Time spent in all parts of the cage (divided into 9 equal squares)
Measures of emotionality recorded for the first 5 minutes only. The following indices of emotionality, as described by Hall and Spyker, were recorded:
 Defecation
 Urination
 Rearing
 Grooming
 Backing

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1)
HISTOPATHOLOGY: Yes (see Table 1)

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, as presented in Table 2.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 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.

Dermal irritation:

effects observed, treatment-related

Description (incidence and severity):

minimal to moderate erosion / ulceration, epidermal hyperplasia and exudate; minimal to mild acute to subacute / chronic inflammation, and; minimal edema in 300 and 1000 mg/kg bw/day dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

300 and 1000 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 weight changes:

effects observed, treatment-related

Description (incidence and severity):

Trend to decreasing mean bw in males at 1000 mg/kg bw/day - not statistically significant, secondary to dermal effects. Mean bw change was statistically decreased in treated males at 1000 mg/kg bw/day during the premating interval from Weeks 1 to 3.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

mean food consumption did not show any statistically significant changes for the treated (including recovery group) male and female animals during the dosing period, compared to controls.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

No adverse test article-related effects on haematology parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No adverse test article-related effects on clinical chemistry analytes.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No definitive test article-related patterns observed in the weekly neurobehavioural examinations between the treated (and recovery group) male and female animals, as compared to controls.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

relative mean spleen weight increased in 1000mg/kg/d males. Considered to be secondary adaptive response to localised dermal effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test article-related macroscopic findings at 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 (300-1000 mg/kg/d)

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No adverse test article-related effects on histopathological: neoplastic parameters.

Details on results:

CLINICAL SIGNS AND MORTALITY
300 and 1000 mg/kg bw/day animals: localized scabbed area on dorsal surface and scabbed area in the thoracic region (related to test site) during dosing period. These were dose-responsive, being most pronounced at the high dose level of 1000 mg/kg bw/day in both males and females. Recovery animals indicated trend towards recovery after cessation of dosing, although these dermal fndings remained evident. [See attached Parental Summary Tables 1-4; and Recovery Summary Table 32]

DERMAL IRRITATION SCORES
Daily dermal scoring (edema, as well as erythema and eschar) during the course of the dosing period in treated male and female animals, revealed dose responsive effects, particularly at 300 and 1000 mg/kg bw/day, with controls being devoid of any significant adverse dermal effects. [See attached Parental Summary Tables 8-11]
The results of the dermal scoring in the animals at 100 mg/kg/day were generally comparable to the controls. Edema in the males at 300 mg/kg/day was limited to very slight effects during the start of dosing, which progressed to slight edema, with a few instances of moderate edema at a little over 5 weeks into dosing. Edema in the males at 1000 mg/kg/day started at very slight effects right after dosing, which became slight edema after about a week of dosing and progressed to moderate edema by 3 weeks of dosing. Edema in the females at 300 mg/kg/day was similar to the males up to the time of mating. At 1000 mg/kg/day, edema also progressed to slight edema by Day 2 of dosing, prior to mating. During gestation, edema was very slight in the 100 mg/kg/day group; it was slight in appearance at 300 mg/kg/day and moderate at 1000 mg/kg/day. During lactation edema was limited to very slight effects at 300 and 1000 mg/kg/day.
The evaluation of erythema and eschar (to be referred to as erythema) showed more significant dermal scoring in the treated animals. Erythema in the males at 300 mg/kg/day was generally very slight starting about a week of dosing and continued during the dosing period (Days 1 to 44). Erythema in the males at 1000 mg/kg/day started with very slight to well-defined effects, and by the first week of dosing progressed from well defined to moderate erythema, with a few instances of severe edema. By three weeks of dosing, most of the males at 1000 mg/kg/day had moderate to severe findings. Erythema in the females at 300 mg/kg/day during the premating period started with very slight findings and some well defined effects about 5 days into dosing and this progressed to well-defined findings by two weeks of treatment until mating. Erythema at 1000 mg/kg/day for females during the premating period started with very slight to well defined findings, which progressed to well defined, moderate, and in a few instances severe erythema by two weeks of dosing, prior to mating.
These localized dermal effects at 300 and 1000 mg/kg/day were considered to be test article related. In the designated recovery animals the dermal scores were similar to the terminal animals, but there was a decreasing trend in severity and incidence after cessation of dosing. [See attached Recovery Summary Table 34]

BODY WEIGHT AND WEIGHT GAIN
There was a trend of decreasing mean body weight in the males at 1000 mg/kg/day, though, the values were not statistically identified. Mean body weight change was statistically decreased in the treated males at 1000 mg/kg/day during the premating interval from Weeks 1 to 3. Mean body weight and body weight gain in the treated females were generally similar to the controls and did not show any definitive treatment related effects over the
dosing period. [See attached Parental Summary Tables 16-27]
In the designated recovery animals, mean body weight at 1000 mg/kg/day was statistically decreased in the males during Weeks 3, 4, 5, 6, and 7, which correlated to the decreases in body weight gain in these animals during weekly study intervals 1-2, 2-3, 3-4, 4-5, and 1-7. Mean body weight at 1000 mg/kg/day continued to be significantly lower than controls during the recovery period; however, the mean body weight gain was comparable to the controls, indicating a reversal of the body weight effects after cessation of dosing. The designated recovery females did not show any statistically significant body weight changes, as compared to the controls. [See attached Recovery Summary Table 35-36]
Effects on body weight and weight gain may have also reflected an adaptive response secondary to test article-related inflammation in treated skin.

HAEMATOLOGY
There were no adverse test article-related effects on haematology parameters. At termination, mean neutrophils tended to higher than expected ranges in all groups, including controls. However, in both males and females, the neutrophils tended to be highest at 1000 mg/kg/day. At recovery, neutrophils remained increased in one of the two females (animal number 400) at 1000 mg/kg/day. A few other statistical differences were observed and considered not meaningful due to the magnitude of change. [See attached Clinical Pathology Summary Table 1]

CLINICAL CHEMISTRY
There were no adverse test article-related effects on clinical chemistry analytes. Mean alanine aminotransferase (ALT) was above typically expected ranges in all groups, including controls at termination, but to noticeably greater magnitude in the females at 1000 mg/kg/day. Aspartate aminotransferase (AST) also tended to increase to a greater magnitude in the females at 1000 mg/kg/day. Globulins tended to increase and were accompanied by decreased albumin to globulin ratios in all treated groups relative to controls, but remained within expected ranges at 100 and 300 mg/kg/day. At recovery, AST, ALT, and the globulins remained mildly to moderately increased in both sexes, but particularly in animal number 348 in the males. This animal also exhibited increased sorbitol dehydrogenase and glucose. A few other statistical differences were observed and considered not meaningful due to the magnitude of change. [See attached Clinical Pathology Summary Table 2]

NEUROBEHAVIOUR
No definitive test article-related patterns observed in the weekly neurobehavioural examinations between the treated male and female animals (100, 300, and 1000 mg/kg bw/day), as compared to controls. Similarly, the designated recovery animals at 1000 mg/kg bw/day did not reveal any definitive changes in the weekly neurobehavioural evaluations that could be attributed to treatment. [See attached Parental Summary Tables 5-7; and Recovery Summary Table 33]

ORGAN WEIGHTS
Possible test article-related organ weight differences at the terminal necropsy were present in the spleen. [See attached Pathology Summary Table 2; Recovery Pathology Summary Table 6]
At the terminal necropsy, the mean spleen weight (relative to body weight) was statistically significantly increased in males at 1000 mg/kg/day versus controls. The increased spleen/body weight ratio may have been associated with the lower mean body weight of males at this dose level compared to controls and/or the increased mean absolute spleen weight that may have reflected increases in extramedullary hematopoiesis noted microscopically.
Additional statistically significant organ weight differences at the terminal necropsy were limited to the increased mean adrenal gland weight (relative to body weight) of females at 1000 mg/kg/day compared to controls. There was no microscopic correlate for this effect, and the relationship to test article administration was unclear.

GROSS PATHOLOGY
Test article-related macroscopic findings were limited to the dose site in the skin. At the terminal necropsy, minimal to moderate abrasion/scab formation was identified in the dose site in the skin in control males and treated animals of both sexes with a dose-related increase in the incidence and/or severity in males at 300 and 1000 mg/kg/day and in females at all dose levels compared to controls. This reflected the test article-related erosion/ulceration noted microscopically in treated skin (see Microscopic section). At the recovery necropsy, the incidence and overall severity of abrasion/scab formation was reduced in animals at 1000 mg/kg/day, indicating partial resolution of this finding over the recovery period.
Other macroscopic findings were infrequent and typical of those commonly identified in rats of this strain and age. [See attached Pathology Summary Table 1; Recovery Pathology Summary Table 5]

HISTOPATHOLOGY: NON-NEOPLASTIC
Test article-related microscopic findings were present in treated skin, and possible test article effects were also noted in the thymus and spleen. Test article-related increases in the incidence and/or severity of microscopic findings in treated skin were present in males at 300 and 1000 mg/kg/day and in females at all dose levels versus controls at the terminal necropsy. These findings were generally present with a dose-related increase in incidence and severity, however, treated skin was not examined in all mid-dose animals (microscopic examination limited to animals with gross lesions). Test article-related microscopic findings in treated skin included minimal to moderate erosion/ulceration, epidermal hyperplasia and exudate, minimal to mild acute to subacute/chronic inflammation, and minimal edema. At the recovery necropsy, test article-related microscopic findings in treated skin in animals at 1000 mg/kg/day were similar, although they occurred at a lower incidence and severity indicating partial resolution of these findings over the recovery period. Minimal to mild cortical lymphoid depletion was noted in the thymus in animals of both sexes at 1000 mg/kg/day at the terminal necropsy while this finding was not identified in control animals. However, notably, there were no statistically significant differences in the mean thymus weight and the thymus was not examined microscopically for all control and treated animals. Thymic lymphoid depletion in animals at 1000 mg/kg/day may have been a secondary finding due to stress associated with the test article-related lesions in treated skin as evidenced by self-mutilation and hypersensitivity to touch noted clinically in males at this dose level. The thymus was not examined microscopically at the recovery necropsy. At the terminal necropsy, there was a slight increase in the incidence and severity of extramedullary hematopoiesis in the spleen in animals at 1000 mg/kg/day versus controls. The severity of splenic extramedullary hematopoiesis was generally increased in both control and treated females, likely due to recent parturition, but the slight increase in the incidence of this finding in animals at 1000 mg/kg bw/day may have also reflected an adaptive response secondary to test article-related inflammation in treated skin. The spleen was not examined microscopically at the recovery necropsy. [See attached Pathology Summary Table 3; Recovery Pathology Summary 7].

CONCENTRATIONS REFERRED TO ABOVE ARE NOMINAL CONCENTRATIONS.

Dose descriptor:

NOAEL

Remarks:

- systemic effects

Effect level:

1 089.75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: NOAEL established for systemic effects on basis that no statistically significant adverse (treatment related) effects were observed at all test doses.

Dose descriptor:

NOAEL

Remarks:

- local effects

Effect level:

111.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

See attached documents

Conclusions:

Based on these data, the systemic no-observable adverse effect level (NOAEL) for repeated dose toxicity is 1089.75 mg/kg bw/day, and the local NOAEL for repeated dose toxicity is 111.25 mg/kg bw/day based on dermal irritation.

Executive summary:

This study was conducted for to evaluate the possible adverse effects of the test article, Fatty acids C18-(unsaturated) lithium salts

, following repeated exposure and to evaluate reversibility, progression, or delayed appearance of any observed changes following a 2-week postdose observation period. The evaluation included systemic toxicity, male and female reproductive performance (gonadal function, estrous cycle, mating behavior, conception, development of the conceptus, and parturition) and neurologic potential in rats. Three treatment groups of ten P (parental) CD®[Crl:CD®(SD)] rats/sex/group were administered the test article at nominal dose levels of 100, 300, or 1000 mg/kg/day at a dose volume of 2.5 mL/kg. Actual dose levels were 111.25; 345.00, and; 1089.75 mg/kg bw/day. One additional group of ten P animals/sex served as the control and received the vehicle, distilled water. The vehicle or test article was administered to all groups via dermal application once daily for approximately 6 hours. The males were dosed for 43 days, beginning 14 days prior to pairing. Dosing of the females began 14 days prior to pairing, through the mating period, up to and including Gestation Day (GD) 19.

Additionally, two groups of five animals/sex/group at 0 (vehicle) or 1000 mg/kg/day were dosed for a total of 43 days and then began a 14-day recovery period. Observations of these animals included clinical signs, neurobehavioral observations, dermal irritation scores, body weights, food consumption, and clinical pathology parameters. At the end of the recovery period, necropsy examinations were performed, organ weights were recorded, and selected tissues were collected and preserved.

Observations of the P animals included clinical signs, neurobehavioral observations, behavioral indices, dermal irritation scores, body weights, and food consumption during the premating/mating, gestation, and lactation periods, clinical pathology parameters, and parturition and litter data. Observations of the offspring (F1) included survival at birth and during lactation, individual pup body weights, and gross abnormalities. At study termination, necropsy examinations were performed on all P animals, organ weights were recorded, and selected tissues were collected and microscopically examined. On LD 4, surviving F1pups were examined externally, euthanized, and discarded.

All animals on study survived to scheduled terminal and recovery necropsies. The most significant clinical observations consisted of dose responsive localized dermal effects at the test site (dorsal surface), consisting of scabbed postdose findings and elevated dermal scores. These findings were most pronounced at 1000 mg/kg/day and were seen most frequently at this dose level. The findings at 300 mg/kg/day were at a lower incidence and with lower dermal scores. Dermal scores and findings at 100 mg/kg/day were comparable to the controls. Mean body weight showed decreasing trends during the course of the study in males at 1000 mg/kg/day. For the remaining animals there were no consistent patterns of adverse effects on body weight or food consumption at the dose levels tested. The results of the neurobehavioral and behavioral evaluations did not reveal any definitive patterns consistent with treatment-related effects.

At termination, there were no adverse test article-related effects on hematology or clinical chemistry parameters evaluated.

The results at scheduled necropsy showed test article-related macroscopic findings that were limited to the dose site in the skin, consisting of minimal to moderate abrasion/scab formation at the dose site in both sexes. At the recovery necropsy, the incidence and overall severity of abrasion/scab formation was reduced at 1000 mg/kg/day, indicating partial resolution of this finding over the recovery period.

Possible test article-related organ weight differences at terminal necropsy included the increased spleen and adrenal gland weights (relative to body weight) of animals at 1000 mg/kg/day. The increased spleen/body weight ratio of males may have been associated with the lower mean body weight at this dose level and, possibly, increases in extramedullary hematopoiesis noted microscopically. There was no microscopic correlate for the increased adrenal gland/body weight ratio of females at this dose level.

Test article-related microscopic findings were present in treated skin at 300 and 1000 mg/kg bw/day, and possible test article effects were also noted in the thymus and spleen at 1000 mg/kg/day.

Test article-related findings in treated skin included an increase in the incidence and/or severity of erosion/ulceration, epidermal hyperplasia and exudate, acute to subacute/chronic inflammation, and edema. At recovery necropsy, test article-related microscopic findings in treated skin in animals at 1000 mg/kg/day were morphologically similar, although they occurred at a lower incidence and severity indicating partial resolution of these findings over the recovery period.

Minimal to mild cortical lymphoid depletion was noted in the thymus in animals of both sexes at 1000 mg/kg/day at the terminal necropsy. However, there were no statistically significant differences in the mean thymus weight. Thymic lymphoid depletion in animals at 1000 mg/kg/day may have been a secondary finding due to stress associated with the test article-related lesions in treated skin. Splenic extramedullary hematopoiesis was slightly increased in both sexes at 1000 mg/kg/day. The increased severity of splenic extramedullary hematopoiesis in control and treated females may have been due to recent parturition, but the slight increase in the incidence of this finding in animals at 1000 mg/kg/day may have also reflected an adaptive response secondary to test article-related inflammation in treated skin.

In conclusion, 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/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.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to or similar to guideline study OECD 422; GLP

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar 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

Sex:

male/female

Details on test animals or test system 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

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 take 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

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.

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).

Duration of treatment / exposure:

Males dosed for at least 43 days, beginning 14 days prior to mating (Groups 1-4).
Dosing of the females beginning 14 days prior to mating and continuing through mating, up to and including GD 19 (Groups 1-4). Another set of animals (Groups 5 and 6) dosed for a total of 43 days before beginning a 14-day (non-treated) recovery period.

Frequency of treatment:

Daily, once per day for 6 hours.

Details on study schedule:

- Age at mating of the mated animals in the study: approximately 10 weeks of age

Remarks:

Doses / Concentrations:
0; 100; 300; 1000 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
0; 111.25; 345.00; 1089.75 mg/kg bw
Basis:
analytical conc.

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/day (water based Fatty acids C18-(unsaturated) lithium salts) and for two weeks at 2106 mg/kg/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/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/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/day) was selected with 10-fold decrease to insure a no effect level, and a nominal mid-dose level of 300 mg/kg/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.

Positive control:

Not included

Parental animals: Observations and 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
- 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:
 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

Oestrous cyclicity (parental animals):

Not determined

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:

Litters were examined as soon as possible after delivery(LD 0) and on LD 4.

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals on day 44 (Groups 1-4) and day 57 (groups 5 and 6)
- Maternal animals: All surviving animals on LD 4 (groups 1-4) and day 57 (groups 5 and 6)

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cranial, thoracic, and abdominal viscera, and recorded number of total implantation scars and number of corpora lutea on each ovary.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring sacrificed on LD 4.
- These animals were subjected to postmortem external examinations.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

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, as presented in Table 2.

Reproductive indices:

Male and female mating index; fertility index and fecundity index.

Females Delivering Litters (%) = (No. females delivering litters / No. females pregnant) x 100

Females with All Stillborn (%) = (No. litters with all stillborn pups / No. females delivering litters) x 100

Females with Stillborn Pups (%) = (No. litters with at least 1 stillborn pup / No. females delivering litters) x 100

Fertility Index - Males (%) = (No. males impregnating a female / No. males paired) x 100

Fertility Index - Females (%) = (No. females pregnant / No. females paired) x 100

Offspring viability 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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

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 weight changes:

effects observed, treatment-related

Description (incidence and severity):

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.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

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.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

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.

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

male/female fertility and fecundity indices and copulatory intervals unaffected by treatment.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
300 and 1000 mg/kg bw/day animals: localized scabbed area on dorsal surface and scabbed area in the thoracic region (related to test site) during dosing period. These were dose-responsive, being most pronounced at the high dose level of 1000 mg/kg bw/day in both males and females. Recovery animals indicated trend towards recovery after cessation of dosing, although these dermal fndings remained evident. [See attached Parental Summary Tables 1-4; and Recovery Summary Table 32]

DERMAL IRRITATION SCORES
Daily dermal scoring (edema, as well as erythema and eschar) during the course of the dosing period in treated male and female animals, revealed dose responsive effects, particularly at 300 and 1000 mg/kg bw/day, with controls being devoid of any significant adverse dermal effects. [See attached Parental Summary Tables 8-11]
The results of the dermal scoring in the animals at 100 mg/kg/day were generally comparable to the controls. Edema in the males at 300 mg/kg/day was limited to very slight effects during the start of dosing, which progressed to slight edema, with a few instances of moderate edema at a little over 5 weeks into dosing. Edema in the males at 1000 mg/kg/day started at very slight effects right after dosing, which became slight edema after about a week of dosing and progressed to moderate edema by 3 weeks of dosing. Edema in the females at 300 mg/kg/day was similar to the males up to the time of mating. At 1000 mg/kg/day, edema also progressed to slight edema by Day 2 of dosing, prior to mating. During gestation, edema was very slight in the 100 mg/kg/day group; it was slight in appearance at 300 mg/kg/day and moderate at 1000 mg/kg/day. During lactation edema was limited to very slight effects at 300 and 1000 mg/kg/day.
The evaluation of erythema and eschar (to be referred to as erythema) showed more significant dermal scoring in the treated animals. Erythema in the males at 300 mg/kg/day was generally very slight starting about a week of dosing and continued during the dosing period (Days 1 to 44). Erythema in the males at 1000 mg/kg/day started with very slight to well-defined effects, and by the first week of dosing progressed from well defined to moderate erythema, with a few instances of severe edema. By three weeks of dosing, most of the males at 1000 mg/kg/day had moderate to severe findings. Erythema in the females at 300 mg/kg/day during the premating period started with very slight findings and some well defined effects about 5 days into dosing and this progressed to well-defined findings by two weeks of treatment until mating. Erythema at 1000 mg/kg/day for females during the premating period started with very slight to well defined findings, which progressed to well defined, moderate, and in a few instances severe erythema by two weeks of dosing, prior to mating.
These localized dermal effects at 300 and 1000 mg/kg/day were considered to be test article related. In the designated recovery animals the dermal scores were similar to the terminal animals, but there was a decreasing trend in severity and incidence after cessation of dosing. [See attached Recovery Summary Table 34]

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
There was a trend of decreasing mean body weight in the males at 1000 mg/kg/day, though, the values were not statistically identified. Mean body weight change was statistically decreased in the treated males at 1000 mg/kg/day during the premating interval from Weeks 1 to 3. Mean body weight and body weight gain in the treated females were generally similar to the controls and did not show any definitive treatment related effects over the
dosing period. [See attached Parental Summary Tables 16-27]
In the designated recovery animals, mean body weight at 1000 mg/kg/day was statistically decreased in the males during Weeks 3, 4, 5, 6, and 7, which correlated to the decreases in body weight gain in these animals during weekly study intervals 1-2, 2-3, 3-4, 4-5, and 1-7. Mean body weight at 1000 mg/kg/day continued to be significantly lower than controls during the recovery period; however, the mean body weight gain was comparable to the controls, indicating a reversal of the body weight effects after cessation of dosing. The designated recovery females did not show any statistically significant body weight changes, as compared to the controls. [See attached Recovery Summary Table 35-36]

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male and female fertility and fecundity indices and copulatory intervals unaffected by treatment. [See attached Reproductive Summary Table 28.]
Natural delivery and Litter Data un affected by treatment. [See attached Reproductive Summary Table 29.]

ORGAN WEIGHTS (PARENTAL ANIMALS)
Possible test article-related organ weight differences at the terminal necropsy were present in the spleen. [See attached Pathology Summary Table 2; Recovery Pathology Summary Table 6]
At the terminal necropsy, the mean spleen weight (relative to body weight) was statistically significantly increased in males at 1000 mg/kg/day versus controls. The increased spleen/body weight ratio may have been associated with the lower mean body weight of males at this dose level compared to controls and/or the increased mean absolute spleen weight that may have reflected increases in extramedullary hematopoiesis noted microscopically.
Additional statistically significant organ weight differences at the terminal necropsy were limited to the increased mean adrenal gland weight (relative to body weight) of females at 1000 mg/kg/day compared to controls. There was no microscopic correlate for this effect, and the relationship to test article administration was unclear.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Test article-related macroscopic findings were limited to the dose site in the skin. At the terminal necropsy, minimal to moderate abrasion/scab formation was identified in the dose site in the skin in control males and treated animals of both sexes with a dose-related increase in the incidence and/or severity in males at 300 and 1000 mg/kg/day and in females at all dose levels compared to controls. This reflected the test article-related erosion/ulceration noted microscopically in treated skin (see Microscopic section). At the recovery necropsy, the incidence and overall severity of abrasion/scab formation was reduced in animals at 1000 mg/kg/day, indicating partial resolution of this finding over the recovery period.
Other macroscopic findings were infrequent and typical of those commonly identified in rats of this strain and age. [See attached Pathology Summary Table 1; Recovery Pathology Summary Table 5]

HISTOPATHOLOGY (PARENTAL ANIMALS) - NON-NEOPLASTIC
Test article-related microscopic findings were present in treated skin, and possible test article effects were also noted in the thymus and spleen. Test article-related increases in the incidence and/or severity of microscopic findings in treated skin were present in males at 300 and 1000 mg/kg/day and in females at all dose levels versus controls at the terminal necropsy. These findings were generally present with a dose-related increase in incidence and severity, however, treated skin was not examined in all mid-dose animals (microscopic examination limited to animals with gross lesions). Test article-related microscopic findings in treated skin included minimal to moderate erosion/ulceration, epidermal hyperplasia and exudate, minimal to mild acute to subacute/chronic inflammation, and minimal edema. At the recovery necropsy, test article-related microscopic findings in treated skin in animals at 1000 mg/kg/day were similar, although they occurred at a lower incidence and severity indicating partial resolution of these findings over the recovery period. Minimal to mild cortical lymphoid depletion was noted in the thymus in animals of both sexes at 1000 mg/kg/day at the terminal necropsy while this finding was not identified in control animals. However, notably, there were no statistically significant differences in the mean thymus weight and the thymus was not examined microscopically for all control and treated animals. Thymic lymphoid depletion in animals at 1000 mg/kg/day may have been a secondary finding due to stress associated with the test article-related lesions in treated skin as evidenced by self-mutilation and hypersensitivity to touch noted clinically in males at this dose level. The thymus was not examined microscopically at the recovery necropsy. At the terminal necropsy, there was a slight increase in the incidence and severity of extramedullary hematopoiesis in the spleen in animals at 1000 mg/kg/day versus controls. The severity of splenic extramedullary hematopoiesis was generally increased in both control and treated females, likely due to recent parturition, but the slight increase in the incidence of this finding in animals at 1000 mg/kg bw/day may have also reflected an adaptive response secondary to test article-related inflammation in treated skin. The spleen was not examined microscopically at the recovery necropsy. [See attached Pathology Summary Table 3; Recovery Pathology Summary 7]

OTHER FINDINGS (PARENTAL ANIMALS)

Dose descriptor:

NOAEL

Effect level:

> 1 089.75 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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.

Dose descriptor:

other: NOAEL non-reproductive systemic effects

Effect level:

345 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: body weight

Dose descriptor:

other: NOAEL local effects

Effect level:

111.25 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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. [See attached Reproductive Summary Tables 30 and 31.]

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

> 1 089.75 mg/kg bw/day

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.

Reproductive effects observed:

not specified

See attached documents.

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/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:

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/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/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/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/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/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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

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

Test material information

Constituent 1

Reference substance name:

Fatty acids C18-(unsaturated) lithium salts

IUPAC Name:

Fatty acids C18-(unsaturated) lithium salts

Constituent 2

Reference substance name:

502962-81-4

Cas Number:

502962-81-4

IUPAC Name:

502962-81-4

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 or test system 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

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)

1

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

2

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: other:

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
- 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: Developmental toxicity

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.