Amines, C10-14-tert-alkyl

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

concentration-driven

Effects on fertility

Description of key information

Reproductive toxicity
One-generation 
The reproductive and developmental NOEL was 250 ppm in rats (OECD TG 415)

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Data waiving:

other justification

Justification for data waiving:

other:

Remarks:

Doses / Concentrations:

Basis:

Reproductive effects observed:

not specified

Reference 2

Endpoint:

two-generation reproductive toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Potential effects of the test material on fertility were evaluated in a one generation reproduction study in rats. There were no treatment-related effects on estrus cycling at any dietary concentration up to and including 1500 ppm as determined by number of estrus stages during the evaluation period or mean cycle length. There were no treatment-related effects on sperm motility, morphology, epididymal sperm count or concentration, testicular sperm count and concentration at any dietary level up to and including 1500 ppm.

Additionally, in a 4-week repeated-exposure inhalation toxicity study testicular weights and testicular as well as uterine histopathology was not altered following exposure to the test material. In 4-week repeated-exposure dermal toxicity study testicular weight and histopathology was not altered following exposure to the test material.

Short description of key information:
Reproductive toxicity
One-generation
The reproductive and developmental NOEL was 250 ppm in rats (OECD TG 415)
BMDL10 511 ppm

Effects on developmental toxicity

Description of key information

Developmental toxicity/teratogenicity
The NOAEL for teratogenicity 45 mg/kg in rats (OECD TG 414)

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:CD (SD)IGS BR VAF/Plus

Details on test animals or test system 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

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

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

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.

Dose descriptor:

NOAEL

Effect level:

5 mg/kg bw/day

Basis for effect level:

other: maternal toxicity

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

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

45 mg/kg bw/day

Species:

rat

Additional information

Presumed pregnant Crl:CD®(SD)IGS BR VAF/Plus® rats were exposed to solutions of the test substance in Neutral Oil 100 N administered percutaneously once daily on days 6 through 20 of presumed gestation (DGs 6 through 20) at dosages of 0 (Vehicle), 5, 15 and 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. The maternal no-observed-adverse-effect-level 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 45mg/kg/day dose group. No developmental effects were seen at any doses that were related to treatment with test material. The developmental NOEL is 45 mg/kg/day.

Toxicity to reproduction: other studies

Link to relevant study records

Reference

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 415; One-Generation Reproductive Study

Deviations:

no

GLP compliance:

yes

Type of method:

in vivo

Species:

rat

Strain:

other: Crl:CD BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories- Kingston, Stone Ridge, New York, USA
- Age at study initiation: (P) 6-7 wks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g
- Fasting period before study: not reported
- Housing: suspended stainless steel cages; 2/cage for the first week of acclimation; 1/cage thereafter
- Use of restrainers for preventing ingestion (if dermal): not applicable- oral study
- Diet (e.g. ad libitum): PMI certified rodent diet 5002M
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 21 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24 degrees C
- Humidity (%): 30 to 70%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hour light / 12 hour dark


IN-LIFE DATES: From: June 2, 1999 To: November 17, 1999

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Treated diets were prepared in increasing order of concentration to prevent contamination of a lower dose with a higher dose. The appropriate amount of test substance was weighed, dissolved in acetone and mixed with approximately 1 kg of untreated feed in a mixer bowl. This mixture was blended in an open Hobart planetary mixer for approximately 15 minutes in a fume hood to evaporate the acetone and to form a uniform premix. The premix was brought to the appropriate final weight with untreated feed and blended for an additional 20 minutes in a Patterson-Kelley cross flow blender.


DIET PREPARATION
- Rate of preparation of diet (frequency): every 2 weeks
- Mixing appropriate amounts with (Type of food): PMI certified rodent diet 5002M
- Storage temperature of food: room temperature


VEHICLE
- Justification for use and choice of vehicle (if other than water): acetone; soluble in acetone not water
- Concentration in vehicle: approximately 1:2 (w/v)
- Amount of vehicle (if gavage): not applicable
- Lot/batch no. (if required): not reported
- Purity: not reported

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples from the top, middle and bottom of the diets at each concentration from the first and second diet preparation, were collected and analyzed for active ingredient to confirm homogeneity. Samples of diets prepared for weeks 2, 6, 10, 14 and 18 were analyzed for active ingredient content to determine proximity to target concentrations. All samples were extracted with hexane, flitered and analyzed by gas chromatography with flame ionization detection. Samples were stored in the freezer until analysis if analysis was not completed on the same day samples arrived.

Duration of treatment / exposure:

1 generation; Parental animals were dosed daily seven days per week. Treatment continued throughout gestation, lactationa dn until terminal necropsy.

Frequency of treatment:

continuous

Duration of test:

four weeks

Remarks:

Doses / Concentrations:
0, 250, 750, 1500 ppm (0, 19.1, 55.6, 107.3 mg/kg/day (males); 0, 21, 62.8, 124.1 mg/kg/day (females))
Basis:
nominal in diet

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Details on study design:

MATING PROCEDURES: Adult female rats were placed individually with an assigned male from the same treatment group, and observed daily until copulation was verified by the presence of a sperm plug in situ, multiple sperm plugs on the cage liner (at least 3), and/or sperm in a vaginal lavage sample. STANDARDIZATION OF LITTERS: Litters were culled to 8 pups (4/sex/litter) on postnatal day 4. PARAMETERS ASSESSED DURING STUDY P AND F1: - Clinical observations: body weight, body weight gain, feed consumption, and clinical signs in parental animals - Estrous cycle: Estrus cycling was evaluated in parental females for three weeks prior to mating. - Sperm examination: Sperm evaluation was performed on all parental males at the time of necropsy. PARAMETERS ASSESSED DURING STUDY F1 AND F2: - Clinical observations and frequency: body weight, body weight gain, feed consumption, and clinical signs in parental animals. - Others: Females were examined daily for vaginal opening beginning at 25 days of age. Males were examined daily for preputial separation beginning at 35 days of age. Body weights were recorded on the day sexual maturation was achieved. OFFSPRING: Postnatal day 0 examinations included - status, sex, weight, external structural abnormalities, and clinical signs of ill health. Sex was also determined at post natal days 4, 7, 14, and 21. During lactation - mortality, morbidity, and obvious indications of a toxic effect performed daily. ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): - Organ weights P and F1: Parental - (females) uterus, ovaries (with oviducts); (males) epididymis (single), caudia epididymis (single), seminal vesicles (with coagulating glands) with their fluids and prostate (as one unit). Offspring - none - Histopathology P and F1: Parental - reproductive organs (vagina, uterus with cervix, ovaries with oviducts in females; testis, epididymis, seminal vesicles with coagulating glands, prostate in males), pituitary gland, stomach and gross lesions for all animals in the 1500 ppm dose group and control group, and all animals found dead or sacrificed during study. In addition, the uterus was examined in the 750 ppm dose group. Reproductive organs were examined in all 250 and 750 dose group animals suspected of reduced fertility. All tissues exhibiting gross pathological changes were examined microscopically. The post-lactation ovary was examined for primordial and growing follicles, as well as the large corpora lutea of lactation. Offspring - none - Histopathology F1 not selected for mating, F2:

Statistics:

The litter (i.e., proportion of pups/litter, or litter mean) was used as the experimental unit for the purpose of statistical evaluation. The level of statistical significance selected was p<0.05. the statistical tests that were used to analyze the parameters studied were: Analysis of Variance (ANOVA), 2xN Chi-square test, and 2XN Kruskal-Wallis nonparametric ANOVA.

Dose descriptor:

NOEL

Effect level:

250 ppm

Sex:

male/female

Basis for effect level:

other: Delayed vaginal opening in females at 750 ppm and 1500 ppm. Delayed preputial separation in males at 1500 ppm. 250 ppm = 19.1 mg/kg/day males; 21.0 mg/kg/day females

Dose descriptor:

BMDL10

Effect level:

511 ppm

Sex:

male/female

Basis for effect level:

other: pup body weight Day 21

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: - Parental data and F1: - Body weight: No body weight effects in parental animals at 250 ppm. Cumulative body weight gain decreased after first week of treatment in males and throughout most of premating period in females at 750 ppm. Decreased mean body weight and cumulative body weight gain during premating period in both sexes at 1500 ppm. No body weight effects on pups at 250 ppm. Decreased mean body weight in pups from postnatal day 4 on at 750 ppm and at all intervals at 1500 ppm. - Food/water consumption: No feed consumption effects in parental animals at 250 ppm or females at 750 ppm. Decreased feed consumption in both sexes at 1500 ppm throughout premating and in males at 750 ppm from week 3 to end of premating period. No effect on feed consumption during gestation or lactation at 750 ppm or less but decreased at 1500 ppm. - Description, severity, time of onset and duration of clinical signs: No clinical signs of systemic toxicity in parental animals of either sex during premating, females during gestation, or females and pups during lactation at any dose level. - Fertility index: No treatment-related effects in males or females at any dose level. - Precoital interval: No treatment-related effects at any dose level. - Duration of gestation: No treatment-related effects at any dose level. - Gestation index: No treatment-related effects at any dose level. - Changes in lactation: No treatment-related effects at any dose level. - Changes in estrus cycles: No treatment-related effects at any dose level. - Effects on sperm: No treatment-related effects at any dose level. - Hematological findings incidence and severity: not evaluated - Clinical biochemistry findings incidence and severity: not evaluated - Mortality: No treatment-related deaths at any dose level. - Gross pathology incidence and severity: No treatment-related effects at any dose level. - Number of implantations: No treatment-related effects at any dose level. - Number of corpora lutea: No treatment-related effects at any dose level. - Ovarian primordial follicle counts: No treatment-related effects at any dose level. - Organ weight changes: No treatment-related effects at any dose level. - Histopathology incidence and severity: No histopathology at any dose level. - Offspring toxicity F1 and F2: No treatment-related effects at any dose level. - Litter size and weights: No treatment-related effects at any dose level. - Sex and sex ratios: No treatment-related effects at any dose level. - Viability index: No treatment-related effects at any dose level. - Post natal survival until weaning: no deaths - Effects on offspring: No treatment-related effects at any dose level. - Postnatal growth, growth rate: No treatment-related effects at any dose level. - Vaginal opening (F) or preputial separation (M): Delayed vaginal opening in females at 750 ppm and 1500 ppm. Delayed preputial separation in males at 1500 ppm.

The exponential 3, polynomial and power models all had p-values for the global goodness of fit at greater

than 0.1, had residuals less than 2 and had a mix of positive and negative values. These models will be

considered good fits. The Hill model had residuals less than 2 and had a mix of positive and negative

values but it could not calculate the global goodness of fit test so it will not be considered a good fit.

Exponential models 2 and 4 had global goodness of fit p-values less than 0.1 and had at least one residual greater than 2 so they are not good fits. Exponential model 5 had residuals less than 2 but it did not have a

global goodness of fit test so will not be considered a good fit. Exponential model 3 was also considered a better model than exponential 5. The linear model was not a good fit by the global goodness of fit test and had residuals greater than 2.

The BMDL values were similar for the BMR of 1 standard deviation and the BMR of 10% relative deviation. The lowest BMDL was 511 ppm for a BMR of 1 standard deviation with a corresponding BMD of 688 ppm test material.

The lowest BMDL was 521 ppm for a BMR of 10% relative deviation with a corresponding BMD of 687 ppm test material.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm Primene™ 81-R Amine for 10 weeks pre-breeding, through breeding, gestation, lactation phases (Wood et al., 2001). Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. At the request of Dow Advanced Materials, an assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed aclear weight of evidence to support the conclusion that delays in puberty onset by Primene™ 81-R Amine are due to decrements in pup body weight secondary to maternal toxicity and thereforedo notwarrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

Conclusions:

Continuous exposure of rats to the test material in the diet through one generation had a NO Observed Effect Level (NOEL) for parental animal toxicity of 250 ppm [19.1 mg/kg/day in males; 21.0 mg/kg/day in females]. The reproductive and developmental NOEL was 250 ppm due to decreased pup weights at both 750 and 1500 ppm and delayed sexual maturation in females at 750 ppm and in both sexes at 1500 ppm.

The lowest BMDL was 511 ppm for a BMR of 1 standard deviation with a corresponding BMD of 688 ppm Primene™ 81-R Amine.

Executive summary:

This study was performed to investigate the effects of the test material on growth, development and reproductive performance in male and female rats through one generation. The test material was administered in the diet at concentrations of 0, 250, 750 or 1500 ppm. These dietary concentrations resulted in consumed doses of 0, 19.1, 55.6, and 107.3 mg/kg/day respectively in males during premating and 21.0, 62.8, and 124.1 mg/kg/day respectively in females during premating.

 Exposure of parental animals (26/sex/dose; non-littermates) began when the animals were approximately six weeks of age. Parental animals were mated after at least ten weeks of exposure to treated diets. Treatment continued throughout gestation, lactation, and until terminal necropsy.

     Body weight, feed consumption, and clinical signs were monitored in parental animals throughout treatment. Estrus cycling was evaluated in adult females for three weeks prior to mating. Parental animals were euthanized and necropsied after weaning of their litters. Sperm evaluation was performed for all adult males at the time of necropsy. Selected tissues (reproductive tissues) were weighed from all adult animals at necropsy. Selected tissues were collected and preserved for histopathology. Histopathologic evaluation was performed for all tissues in control and high dose animals, and animals found dead or sacrificed during the course of the study. The uteri were examined in all dose levels. Reproductive tissues were also examined in all animals suspected of reduced fertility.

     Survival and growth of offspring were monitored throughout lactation. Litters were culled to 8 pups (4/sex/litter where possible) on postnatal day 4 (PND4). Stillborn pups, pups that died during lactation, and pups euthanized at weaning (PND21) were grossly examined (except for pups chosen for sexual maturation evaluation, which were discarded without gross examination).

     There were no treatment-related deaths or clinical signs of systemic toxicity in parental animals of either sex during premating, females during gestation, or females and pups during lactation at any dietary level. There were no treatment-related effects on body weight in parental animals at 250 ppm. At 1500 ppm, a treatment-related decrease in mean body weight and cumulative body weight gain was evident in parental animals (both sexes) throughout the premating period. Mean body weight and body weight gain in females during both gestation and lactation at this level were also decreased. At 750 ppm, cumulative body weight gain was decreased after the first week of treatment in males and throughout most of the premating period in females. 

     There were no treatment-related effects on premating feed consumption in either sex at 250 ppm or in females at 750 ppm. Feed consumption was decreased in Group 4 (1500 ppm) males and females throughout the premating period and Group 3 (750 ppm) males from week 3 until the end of the premating period.  There was no effect on feed consumption during either gestation or lactation at dietary levels up to and including 750 ppm. At 1500 ppm, feed consumption during gestation and lactation was decreased at all intervals.

     There were no treatment-related effects on estrus cycling at any dietary concentration, as determined by number of estrus stages during the evaluation period or mean cycle length. There were no treatment-related effects on sperm motility, morphology, epididymal sperm count or concentration, or testicular sperm count and concentration at any dietary level.

     There were no treatment-related effects on any endpoint of mating or fertility at any dietary concentration. There were no treatment-related effects on gestation index or gestation length. On PND0, the number of pups per litter in any treatment group was not statistically different from controls. There were no treatment-related deaths during delivery and there were no litters entirely stillborn. There were no treatment-related effects on the mean number of implantation sites or post-implantation loss at any dietary concentration. There were no treatment-related effects on offspring viability or the ratio of male to female pups. 

     There were no treatment-related effects on offspring body weight at 250 ppm. Mean body weight was decreased at all intervals in pups at 1500 ppm and from PND4 on in pups at 750 ppm. There were no treatment-related malformations or variations observed in offspring at necropsy in any treatment group. There were no treatment-related effects on sexual maturation in males up to and including 750 ppm or in females at 250 ppm. The mean age of preputial separation was delayed in males at 1500 ppm. The mean age of vaginal patency was delayed in females at 750 and 1500 ppm.

     There were no treatment-related gross findings in parental animals. There was no effect on the primordial follicle or corpora lutea counts in the high dose females. There were no treatment-related effects on absolute or relative organ weights in either sex at concentrations up to and including 1500 ppm. There were no treatment-related microscopic changes in parental animals (including reproductive tissues) at dietary levels up to and including 1500 ppm. 

In a one-generation reproductive toxicity study, groups of 26 male and female rats were administered diets containing 0, 250, 750, or 1500 ppm test material for 10 weeks pre-breeding, through breeding, gestation, lactation phases. Evaluation of one pup/sex/litter for markers of puberty onset revealed a delay in preputial separation for males in the 1500 ppm group and vaginal patency in females in the 750 and 1500 ppm groups. An assessment of these data and their impact on EU classification for reproductive and/or developmental toxicity was conducted. Detailed evaluation of the study data revealed a clear weight of evidence to support the conclusion that delays in puberty onset by the test material are due to decrements in pup body weight secondary to maternal toxicity and thereforedo not warrant reproductive or developmental classification. This conclusion is based upon 1) decrements in pup body weight at weaning causing delays in the age of puberty onset, 2) the fact that both male and female pups had delays in puberty onset as opposed to advancement in one sex and delay in the other, and 3) the lack of an effect in other endocrine responsive end points.

A benchmark dose (BMD) analysis of relevant toxicity data for primene was conducted in order to establish BMD and BMDL (lower limit of the 95% confidence interval on the BMD) values. The U.S. EPA’s Benchmark Dose Software (BMDS) was used for the analysis (U.S. EPA 2009). Data from a one-generation reproductive toxicity study in rats was used (Wood et al., 2001). The exposure doses were 0, 250, 750, and 1500 ppm of test material.

The key endpoint evaluated was pup body weight. There was a significant effect on pup body weights at the highest dose of 1500 ppm and at the mid-dose (750 ppm). Delayed sexual maturation was also considered but was secondary to the body weight effects so this endpoint was not modeled.

The exponential 3, polynomial and power models all had p-values for the global goodness of fit at greater than 0.1, had residuals less than 2 and had a mix of positive and negative values. These models were considered good fits. The lowest BMDL was 511 ppm for a BMR of 1 standard deviation with a corresponding BMD of 688 ppm test material.

 

Additional information

This study was performed to investigate the effects of the test material on growth, development and reproductive performance in male and female rats through one generation. The test material was administered in the diet at concentrations of 0, 250, 750 or 1500 ppm. These dietary concentrations resulted in consumed doses of 0, 19.1, 55.6, and 107.3 mg/kg/day respectively in males during premating and 21.0, 62.8, and 124.1 mg/kg/day respectively in females during premating. Continuous exposure of rats to test material in the diet through one generation had a No Observed Effect Level (NOEL) for parental animal toxicity of 250 ppm [19.1 mg/kg/day in males; 21.0 mg/kg/day in females]. The reproductive and developmental NOEL was 250 ppm due to decreased pup body weights at both 750 and 1500 ppm and delayed sexual maturation in females at 750 ppm and in both sexes at 1500 ppm. Detailed evaluation of the study data revealed a clear weight of evidence to support the conclusion that the delays in puberty onset by the test material are due to decrements in pup body weight secondary to maternal toxicity.

Mode of Action Analysis / Human Relevance Framework

Not applicable, no reprodevelopmental effects observed

Justification for classification or non-classification

No developmental effects were seen at any doses that were related to treatment with the test material in a percutaneous pre-natal developmental study.

The test material is not classified as a reproductive toxicant. Detailed evaluation of the study data revealed a clear weight of evidence to support the conclusion that delays in puberty onset by the test material are due to decrements in pup body weight secondary to maternal toxicity and therefore do not warrant reproductive or developmental classification.