Registration Dossier

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: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:CD (SD)IGS BR VAF/Plus
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan, USA
- Age at study initiation:62 days, at receipt; breeder male- 77 days, at receipt
- Weight at study initiation: 196-256 g; breeder male- 294-446 g
- Fasting period before study:
- Housing: 1/cage in stainless steel cages
- Diet (e.g. ad libitum): Certified rodent diet #5002
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 53.1-71.7 degrees C
- Humidity (%): 29.3-73.3%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark


IN-LIFE DATES: From: September 30, 2002 To: October 24, 2002

Administration / exposure

Route of administration:
dermal
Vehicle:
other: Neutral oil 100 N
Details on exposure:
TEST SITE
- Area of exposure: backs; 5x7 cm extending from the shoulders to approximately 2 cm anterior to the hip joints and was approximately 5 cm wide (extending ventrolaterally from the dorsal midline approximately 2.5 cm on each side)
- % coverage: not reported
- Type of wrap if used: Elizabethan collar and Lomir jacket
- Time intervals for shavings or clipplings: any regrowth of hair was shaved as needed throughout the study


REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed with warm water (deionzed water) and then blotted dry with a clean, disposable wipe (Nu-Gauze)
- Time after start of exposure: 6 hours


TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 2.0 mL/kg
- Concentration (if solution): 2.5, 7.5, 22.5 mg/L
- Constant volume or concentration used: yes; adjusted daily for body weight changes
- For solids, paste formed: not applicable


VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance was not solube in water but was soluble in the oil
- Amount(s) applied (volume or weight with unit): 2.0 mg/L
- Concentration (if solution): 2.5, 7.5, 22.5 mg/L
- Lot/batch no. (if required): 2002-148
- Purity: not reported


USE OF RESTRAINERS FOR PREVENTING INGESTION: yes; Elizabethan collars and Lomir jackets with an insert to prevent oral ingestion
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were sent for analysis and stored at room temperature. GC/FID was used to measure the Amines, C12-C14 tert-alkyl concentration in these samples. After receipt, the samples were further diluted in tetrahydrofuran. A stock solution of the test material in Neutral oil was prepared as follows: 0.7865 g of test substance in 22.2767 g of oil. To prepare the dilute standard solution, a serial dilution was done.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 in male rat's cage
- Length of cohabitation: 5 days
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. not reported
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- Any other deviations from standard protocol: none
Duration of treatment / exposure:
days 6 - 20 of presumed gestation
Frequency of treatment:
once daily
Duration of test:
21 days
No. of animals per sex per dose:
25
Control animals:
other: 0 (Sham Control), 0 (Vehicle Control)
Details on study design:
- Dose selection rationale: based on dosage range finder
- Rationale for animal assignment (if not random): computer randomization

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice/day for viability and weekly for general appearance and before application and following rinsing during the dosage period and on the day of sacrifice for effects of the test substance, abortions, premature deliveries, and deaths
- Cage side observations checked were included: viability and general appearance, effects of the test substance, abortions, premature deliveries, and deaths


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before application and following rinsing during the dosage period and on the day of scheduled sacrifice.


BODY WEIGHT: Yes
- Time schedule for examinations: DG 6-20


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined : Yes; recorded on Day 0, daily during the dosage period and on the day of sacrifice
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: thoracic, abdominal, and pelvic viscera, uteri and ovaries, number and distribution of corpora lutea, placentae


OTHER: Before the first daily application, and at 24-hr intervals each day thereafter, each skin site was observed for signs of skin irritaion and graded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: placentae were examined for size, color and shape
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter
Statistics:
Clinical observations and other proportional data (number of dams with resorptions, fetal alterations, etc.) were analyzed using the Variance Test Homogeneity of the Binomial Distribution. Continuous data (e.g. maternal body weights, body weight changes, feed consumption values and litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data and fetal ossification site data) were analyzed using Bartlett's Test of Homogeneity of Variances and the Analysis of Variance, when appropriate [i.e., Bartlett's Test was not significant (p>0.001)]. If the Analysis of Variance was significant (p£0.05), Dunnett's Test was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate [i.e., Barlett's Test was significant (p£0.001)], the Kruskal-Wallis Test was used (£75% ties). In cases where the Kruskal-Wallis Test was statistically significant (p£0.05), Dunn's Method of Multiple Comparisons was used to identify the statistical significance of the individual groups. If there were greater than 75% ties, Fisher's Exact Test was used to analyze the data. Count data obtained at Caesarean-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.
Indices:
The litter averages for corpora lutea, implantations, litter
sizes, live fetuses, fetal body weights, early and late resorptions, percent resorbed
conceptuses, and percent live male fetuses
Historical control data:
Historical data were provided for the period of January 2000 to January 2002 from the testing laboratory for the following:
summary of reproductive indices; summary of maternal necropsy observations; summary of fetal gross external alterations; summary of fetal soft tissue alterations; summary of fetal skeletal alterations; summary of fetal ossification sites

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
MATERNAL TOXIC EFFECTS BY DOSE LEVEL:
- Mortality and day of death: The number of rats (eight) sacrificed for humane reasons due to adverse clinical observations (excessive vocalization, hyperactivity, and gasping) occurring immediately following test substance application in the 45 mg/kg/day dosage group was significantly increased (p£0.05) as compared to the sham control and vehicle control groups. These signs were also observed in rats that were not sacrificed; however, the extent of and the duration of the vocalization and hyperactivity did not justify sacrifice. All other rats survived until scheduled sacrifice.
8 at 45 mg/kg/day; DG 10 (2), DG 11 (3), DG 12 (2), DG 13 (1)
- Number pregnant per dose level:
Dosage Group 0 (Sham) 0 (Veh) 5 15 45 (mg/kg/day)
Rats Tested 25 25 25 25 25
Pregnant 25(100) 24(96) 23(92) 23(92) 25(100)
Humane Sac. 0(0) 0(0) 0(0) 0(0) 8(32)
Rats Pregnant and Caesarean-Sectioned on Day 21 of Gestation 25 24 23 23 17
Average Maternal Body Wt. Gain (DG6-21): +145.3 +136.5 +135 +129 +109
±18.6 ±18.8 ±18.0 ±9.4 ±24.6
Absolute Maternal Feed Consumption (DG6-21; g/day): 29.9 27.6 26.5 26.4 25.2
±2.0 ±2.2 ±2.2 ±1.6 ±2.4
Corpora Lutea 16.7 16.5 16.2 16.8 15.4
±2.5 ±2.9 ±1.9 ±2.8 ±1.8
Implantations 14.6 14.4 14.5 14.3 14.0
±2.0 ±1.7 ±1.6 ±1.4 ±1.3
Resorptions 0.4 0.7 0.6 0.5 0.6
±0.5 ±1.6 ±0.8 ±1.0 ±0.8
Early Resorptions 9 17 15 10 11
0.4±0.5 0.7±1.6 0.6±0.8 0.4±1.0 0.6±0.8
Late Resorptions 1 0 0 1 0
0.0±0.2 0.0±0.0 0.0±0.0 0.0±0.2 0.0±0.0
- Description, severity, time of onset and duration of clinical signs: The number of rats (eight) sacrificed for humane reasons due to adverse clinical observations (excessive vocalization, hyperactivity, and gasping) occurring immediately following test substance application in the 45 mg/kg/day dosage group was significantly increased (p£0.05) as compared to the sham control and vehicle control groups. These signs were also observed in rats that were not sacrificed; however, the extent of and the duration of the vocalization and hyperactivity did not justify sacrifice. All other rats survived until scheduled sacrifice.

Additional clinical signs observed in the rats sacrificed included chromodacryorrhea, chromorhinorrhea, grade 1 and 2 flaking and grade 1 erythema and edema, brown discoloration of the skin and localized alopecia (limbs).

The number of rats in the vehicle control group with chromodacryorrhea, chromorhinorrhea and grade 1 erythema (four rats) was increased or significantly increased (p£0.05) as compared to the sham control group values. Since chromodacryorrhea and chromorhinorrhea were seen in the sham control group, these signs were considered related to the restraint procedure as well as the vehicle.

All other clinical observations in the vehicle control group were considered unrelated to the vehicle because: 1) they occurred at similar incidence to the sham control group values or 2) they occurred in only one or two rats. These observations included localized alopecia (head, limbs and underside), a scab at the base of the tail and at the administration site, a red substance on the vagina, soft or liquid feces, no fees and grade 1 flaking.

Test substance related increases or significant increases (p£0.05) in the number or rats with hyperactivity, vocalization, gasping and brown discoloration of the skin were observed in the 15 and 45 mg/kg/day dosage groups, as compared to the sham control group and/or vehicle control group values. Signs of hyperactivity and vocalization were observed in rats that were not sacrificed; however, the extent of and duration of the vocalization and hyperactivity did not justify sacrifice.

The number of rats in the 5, 15, 45 mg/kg/day dosage groups with chromodacryorrhea and chromorhinorrhea was significantly increased (p£0.05) as compared to the sham control group values but not the vehicle control group. These observations were therefore considered vehicle related.

All other clinical observations were considered unrelated to the vehicle or test substance.

- Skin Reactions: The number of rats with localized alopecia on the head was significantly increased (p£0.05) in all treated groups, as compared to the sham control group and vehicle control group values. These increases were not considered test substance related because the number of rats affected did not increase in a dosage dependent manner.

The number of rats in the 5 mg/kg/day dose group with grade 1 erythema and scabs at the administration site were comparable to the vehicle control group values. These observations were therefore considered vehicle related.

Test substance related significant increases (p£0.05) in the number of rats with grade 1 flaking, edema and erythema, grade 2 flaking and scabs at the administration site were observed in the 15 and 45 mg/kg/day dose groups, as compared to the sham and vehicle control groups.

A test substance-related ulceration at the site of administration and significant increases (p£0.05) in the number of rats with grade 2 erythema and edema, grade 3 flaking occurred in the 45 mg/kg/day dose group. Although the ulceration was only observed in one rat, it is considered test substance related because it was observed in a rat that had numerous skin irritation signs.

All other skin reactions were considered unrelated to the vehicle or test substance.

- Gross pathology incidence and severity: Clinical signs observed persistently during the dosage period were confirmed at necropsy; no additional gross lesions were identified.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
FETAL DATA:
- Dosage Group (mg/kg/day) 0 (Sham) 0 (Veh) 5 15 45
Rats Tested 25 25 25 25 25
Pregnant 25 (100) 24 (96) 23 (92) 23 (92) 25 (100)
Litter Sizes 14.2 13.7 13.9 13.8 13.4
± 2.0 ± 2.4 ± 1.8 ± 1.4 ± 1.9
L ive Fetuses 354 328 319 317 228
14.2±2 13.7±2.4 13.9±1.8 13.8±1.4 13.4±1.9
Dead Fetuses 0 0 0 0 0
Live Male Fetuses: 179 159 169 163 118
% Live Male Fetuses/ Litter: 50.8 47.3 53.2 51.4 51.9
±15.8 ±15.3 ±13.2 ±13.2 ±9.8
Live Fetal Body Wts (grams)/ Litter: 5.49 5.31 5.40 5.41 5.23
±0.24 ±0.29 ±0.28 ±0.29 ±0.38
Male Fetuses 5.65 5.44 5.55 5.58 5.30
±0.30 ±0.30 ±0.27 ±0.28 ±0.46
Female Fetuses 5.32 5.20 5.23 5.23 5.12
±0.22 ±0.27 ±0.28 ±0.28 ±0.37

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
On the basis of these data, the maternal no-observable-adverse-effect-level (NOAEL) of the test material is 5 mg/kg/day. Adverse clinical observations, skin reactions and reductions in body weights and feed consumption were observed in the 15 and/or 45 mg/kg/day dose groups. An increase in the number of rats sacrificed for humane reasons was observed in the 45 mg/kg/day dose group.
No developmental effects were seen at any dose that were related to treatment with test material. The developmental NOEL is 45 mg/kg/day.
Executive summary:

One hundred twenty-five presumed pregnant Crl:CD (SD)IGS BR VAF/Plus rats were randomly assigned to five dosage groups (Groups 1 through V), 25 rats per group. Solutions of the test substance in Neutral Oil 100 N, or the vehicle, Neutral Oil 100 N, were administered percutaneously once daily to rats in Groups II through V on days 6 through 20 of presumed gestation (DGs 6 through 20) at dosages of 0 (vehicle), 5, 15 and 45 mg/kg/day. The dosage volume was 2.0 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately before application of the test substance. Rats assigned to Group I were not administered the test substance or vehicle; sham dosing procedures were completed each day with an empty syringe.

Viabilities, clinical observations, skin reaction grading, body weights and feed consumption values were recorded. All surviving rats were sacrificed on DG 21 and Caesarean-sectioned, and a gross necropsy of the thoracic, abdominal and pelvic viscera was performed. Uteri of apparently nonpregnant dams were examined to confirm the absence of implantation sites. Tissue with gross lesions were retained.

The fetuses were weighed and examined for gross external alterations and sex. Approximately on-half of the fetuses in each litter were examined fresh for soft tissue alterations; the remaining fetuses (approximately one-half of the fetuses in each litter) were examined for skeletal alterations after staining.

The number of rats (eight) sacrificed for humane reasons due to adverse clinical observations (excessive vocalization, hyperactivity and gasping) occurring immediately following test substance application in the 45 mg/kg/day dosage group was significantly increased as compared to the sham control and vehicle control groups. These signs were also observed in rats that were not sacrificced; however, the extent of and the duration of the vocaliztion and hyperactivity did not justify sacrifice. All other rats survived until scheduled sacrifice.

Test substance related increases or significant increases in the number of rats with hyperactivity, vocalization, gasping and brown discoloration of the skin occurred in the 15 and 45 mg/kg/day dosage groups. Test substance related significant increases in the number of rats with grade 1 flaking, grade 1 edema and erythema, grade 2 flaking and scabs at the administration site occurred in the 15 and 45 mg/kg/day dosage groups and grade 2 erythema and edema and grade 3 flaking occurred in the 45 mg/kg/day dosage group. An ulceration at the administration site was observed in the 45 mg/kg/day dosage group. No maternal gross lesions occurred at necropsy.

Body weight gains were significantly reduced in the 15 and 45 mg/kg/day dosage groups, as compared to the sham control group and/or vehicle control group values for the entire dosage period (calculated as DGs 6 to 21), for the entire gestation period (dgs 0 to 21) and at numerous tabulated intervals during the dosage period. Test substance related significant reductions in maternal body weights occurred on DGs 10 to 21 in the 45 mg/kg/day dosage group.

Gravid uterine weights were comparable among the test material treated groups as well as the control groups. Corrected maternal body weight gains for DGs 6 to 21C and 0 to 21C (21C = DG 21 body weight minus the gravid uterine weight) were significantly reduced, as compared to the sham control group and/or vehicle control group values, in the 15 and 45 mg/kg/day dosage groups.

Absolute (g/day) feed consumption values were significantly reduced in the 45 mg/kg/day dosage group, as compared to the sham control group and the vehicle control group for the entire dosage period (calculated as DGs 6 to 21). These values were also significantly reduced, as compared to the sham control group and/or the vehicle control group at each tabulated interval during the dosage period.

No Caesarean-sectioning or litter parameters were affected by dosages of the vehicle or test substance as high as 45 mg/kg/day.

No fetal gross external, soft tissue or skeletal alterations (malformations or variations) were caused by dosages of the test substance as high as 45 mg/kg/day. All alterations were considered unrelated to the test substance because: 1)they were not dosage dependent; 2)the litter incidence was not significantly different from either control group value; and/or 3) they were within the ranges observed historically at the Testing Facility.

On the basis of these data, the maternal no observed adverse effect level (NOAEL) is 5 mg/kg/day. Adverse clinical observations, skin reactions and reductions in body weights and feed consumption were observed in the 15 and/or 45 mg/kg/day dosage groups. An increase in the number of rats sacrificed for humane reasons was observed in the 45 mg/kg/day dosage group. No developmental effects were see at any dose that were related to the treatment with Amines, C12-C14 tert-alkyl. The developmental NOEL is 45 mg/kg/day.