Registration Dossier

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

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

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.

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.

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.