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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
40-51 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: valid without restriction; GLP guideline study conducted according to OECD 421 and US EPA OPPTS 870.3550 guidelines

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA OPPTS 870.3550
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Test Substance: 2,2,4-Trimethyl-1,3-Pentanediol Diisobutyrate
- Synonym: TXIB
- Physical State and Appearance: Liquid, Clear colorless
- Source of Test Substance: Eastman Chemical Company, Kingsport, TN
- Purity: 99.0%
- Analytical laboratory: purity analysis was conducted by the Chemicals Quality Services Laboratory at Eastman Kodak Company
- Stability: Based on purity analysis, determined by gas chromatography with FID at the start and end of the study, test material is considered stable.
-Confirmation of identity: determined by GC/MS

Test animals

Species:
rat
Strain:
other: Sprague-Dawley rats (Crl:CD(SD)IGS BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS:
-Source: Charles River Laboratories (Kingston, NY)
-Condition at receipt: animals were isolated upon arrival and were judged healthy prior to testing
-Acclimation period: 5 days
-Age at Study Initiation: 61 days
-Weight at Study Initiation: 294.55 ± 12.07g (males) and 213.79 ± 8.30g (females)
-Method of Animal Identification: uniquely numbered metal ear tag
-Method of Animal Distribution: Animals were culled from the stock population based on body weight and randomly assigned to each test group using a stratified randomization program. Variation among body weights of individuals did not vary more than 20% from the mean.
-Housing: Starting at study initiation and continuing throughout premating, animals were housed singly in suspended, stainless-steel wire mesh cages. During mating, animals were housed one male with one female. Following mating, males were housed singly. On or about the 19th day of gestation, solid bottom pans containing bedding material for nesting were put in the cages of the female rats. Cages were washed once a week and absorbent paper under the cages was changed daily.
-Diet: Certified Rodent Diet [Purina Rodent Chow #5002, meal (PMI Feed Inc. Richmond, IN)] was available ad libitum. Feed containers were cleaned and refilled at least weekly.
-Water: city water (Monroe County NY Water Authority) was supplied ad libitum through an automatic watering system during the premating, mating, and gestation periods. During lactation, females were provided with water in glass bottles with sipper tubes while they were housed with nesting pans in their cages. Water was analyzed on a semiannual basis and analysis reports maintained on file with the testing laboratory.

ENVIRONMENTAL CONDITIONS:
-Temperature (°C): 21.8-25.6
-Relative Humidity: 31.9-56%
-Air changes per hour: no information
-Photoperiod (hrs dark/ hrs light): 12 hours dark/light

IN-LIFE DATES:
-Study initiation: January 25, 2001
-Experimental Start Date: January 29, 2001
-Duration of Dosing: Male rats were treated from the beginning of the premating period to the final treatment for the female rats and received 51 doses over 51 days. Females were treated from the beginning of the premating period to postpartum Day 4 for a total of 40 to 51 treatments.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: Certified Rodent Diet [Purina Rodent Chow #5002, meal]
Details on exposure:
All male rats were allowed unlimited access to feed over 51 days starting on the first day of premating; all males were euthanized the following day. All female rats were similarly treated for 40-51 days; all female rats were euthanized on lactation day 4. The animals were provided diets containing 0, 1.5, 4.5, or 15.0 mg of the test substance per gram of feed ad libitum until they were euthanized. These dietary concentrations produced dose levels of approximately 0, 91, 276 and 905 mg/kg bwt/day for male rats and 0, 120, 359 and 1135 mg/kg bwt/day for female rats. The test substance was mixed with certified ground chow to yield concentrations of 1.5, 4.5, and 15.0 mg/g. Test substance in vehicle mixtures were prepared twice and used within 35 days based on the stability of the mixture. The control animals were given the basal diet. The test material was administered to 12 rats/sex/dose group; animals were allowed ad libitum access to feed seven days per week including holidays.
Details on mating procedure:
Females were mated 1:1 with males of the same group for 1-8 days. Gestation day 0 was defined when a copulatory plug or sperm in a vaginal smear was present. Following copulation, the males and females were housed individually until study termination. Clinical observations were performed on a daily basis.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test substance in test diets was determined by repeated analysis of the 15.0 and 1.5 mg/g mixtures of the test substance. Mixtures were analyzed using GC/FID 0, 3, 7, 9, 21, and 35 days after test mixture preparation. Concentrations of the test substance were 15.5 ± 0.20, and 1.54 ± 0.012 mg/g (mean ± SD) prior to storage, and 14.7 ± 0.16 and 1.42 ± 0.019 mg/g after 35 days of storage, indicating the test substance was stable for at least 35 days in feed.

Homogeneity of the test diets was evaluated by measuring the concentration of the test substance at three levels (top, middle, and bottom of the container) for the 15.0, 4.5, and 1.5 mg/g mixtures of the test substance. For the 15.0 mg/g mixture, the analytical concentrations of the test substance in the top, middle, and bottom layers varied between 14.2 and 15.3 mg/g ± 0.314. For the 4.5 mg/g mixture, the analytical concentrations of the test substance in the top, middle, and bottom layers varied between 4.43 and 4.93 mg/g ± 0.162. For the 1.5 mg/g mixture, the analytical concentrations of the test substance in the top, middle, and bottom layers varied between 1.47 and 1.61 mg/g ± 0.035. Based on these results, the preparations were considered to be homogeneous.

The concentration of the test substance in each batch of test diet was determined by GC/FID analysis prior to use. The mean concentrations of the test substance in batch 1 were 98.0, 102.4, and 102.7% of the target concentrations of 15, 4.5, and 1.5 mg/g, respectively. The mean concentrations of the test substance in batch 2 were 102.0, 100.4, and 99.3% of the target concentrations of 15, 4.5, and 1.5 mg/g, respectively.
Duration of treatment / exposure:
40-51 days for females; 51 days for males
Frequency of treatment:
ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/g
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.5 mg/g
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
4.5 mg/g
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
15.0 mg/g
Basis:
nominal in diet
No. of animals per sex per dose:
12 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale – Dose levels were selected by the Sponsor based on the data from a previous repeated-dose oral toxicity study.

The study was conducted in 4 phases: premating (14 days); mating (1-8 days); pregnancy (21-23 days); and early lactation (4-5 days). Females were treated from the beginning of the premating period to lactation day 4 for a total of 40-51 treatments. Females that delivered a litter and their offspring were sacrificed on days 4 or 5 postpartum. Females that showed evidence of mating but did not deliver were sacrificed on gestation day 23. All male rats were treated from the beginning of the premating period to the final treatment of the female rats for a total of 51 days. Dose levels were 0, 1.5, 4.5, or 15.0 mg/g; animals were exposed to control or test diets 7 days a week.

Examinations

Parental animals: Observations and examinations:
CLINICAL SIGNS: Clinical observations were performed on a daily basis.

BODY WEIGHT: collected on Days 0, 7 and 14 premating and on Days 0, 7, 14, 20 of gestation and on Days 0 and 4 post-partum for females; males were weighed on Days 0, 7, 14, 21, 28, 35, 42, and 49. For the adult male rats, terminal body weights were collected following exsanguination. Terminal body weights were not collected for adult females.

FEED CONSUMPTION: Feeders were weighed on Days 0, 7, and 14 (premating), on gestation days 0, 7, 14, and 20, and on lactation days 0 and 4 for females; determined on Days 0, 7, 14, 35, 42, and 49 for males. Feed consumption was not determined during the mating period.

MATING SUCCESS: Females in mated pairs were examined for up to 8 days to verify mating had occurred. The morning a vaginal plug or sperm was observed was considered Day 0 of gestation.

PARTURITION: Dams were observed for the beginning of parturition and this day was used to calculate the gestation period; the day that the dams had completely finished delivering was considered lactation day 0.
Sperm parameters (parental animals):
MOTILITY EVALUATION: At necropsy, motility of sperm from the right epididymis was determined. The epididymis was placed in a petri dish containing 1% bovine serum albumin in phosphate buffered saline warmed to ~38°C. The epididymis was pierced three times with a scapel blade followed by a period of at least 3 minutes to allow the sperm to swim out. A sample of the mixture in the petri dish was then loaded into a warmed stage of a Hamilton Thorne IVOS automated sperm analyzer. Five fields were automatically selected by the analyzer and each motion image was recorded and stored in the optical disk. The images were subsequently analyzed and the percent motility was determined for each animal.

TESTICULAR SPERMATID HEAD COUNTS: Homogenization-resistant spermatid head counts were performed on the frozen left testes. After the tissue was thawed, the tunica was removed from the testis; the tissue was weighed, homogenized, and then vortexed. A 100 µL sample was transferred to a reaction vial containing a dye that uniquely stains the head of the sperm. A sample of stained sperm was placed into a 20 µm deep glass slide which was loaded into a Hamilton Thorne IVOS automated sperm analyzer. Twenty fields were automatically selected by the analyzer for each animal and total sperm counts were determined.

EPIDIDYMAL SPERMATOZOAN COUNTS: Homogenization-resistant spermatozoan counts were performed on the frozen left epididymides. The epididymal tissue was processed in a similar manner as with the testicular tissue, except that the caudal left epididymis was used.
Litter observations:
All pups were examined as soon as possible after birth to determine the number of viable and stillborn pups. Live pups were sexed. Litters were examined for abnormalities on lactation days 0 through 4 and live pups were weighed as a litter on lactation days 0 and 4.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Necropsies and gross examinations were performed on all animals. Females that failed to deliver were euthanized on Day 23 or 24 of gestation and the uteri were examined for implantation sites. Dams that littered were examined for implantation sites and corpora lutea at the time of necropsy. The following tissues were collected from the adult animals and fixed in 10% buffered formalin: ovaries, vagina, uterus, Fallopian tubes, male accessory sex glands, and gross lesions. After motility analysis, the right testis and right epididymis were fixed in Bouin’s fixative; after approximately 24 hours, these tissues were rinsed twice with 50% ethyl alcohol and then stored in 70% ethyl alcohol. For long-term storage, the right testis and epididymis were stored in 10% buffered formalin. The left testis and epidiymis were placed into individual containers, frozen with dry ice, and stored at -70°C.

ORGAN WEIGHTS: For adult male rats, the testes and epididymides were weighed. Paired organs were weighed together.

HISTOPATHOLOGY: For the control and high-dose groups, the ovaries, right testis, right epididymis, and collected gross lesions were embedded in paraffin and sectioned at 4 µm. The resulting tissue sections were stained with hematoxylin and eosin (H&E) and examined for histopathology. In addition, duplicate tissue sections of the testicular tissues were prepared and stained with the periodic acid-Schiff procedure (PAS). Collected gross lesions from the low and mid-concentration groups were also examined microscopically.
Statistics:
Mean values were calculated for adult body weight and body weight change, male and female pup body weight and body weight change, feed consumption, feed utilization, organ weights, organ-to-body weight ratios, gestation duration, fertility index, fecundity index, precoital interval, implantation counts, post-implantation loss, litter size, and pup percent survival.

Homogeneity of adult body weight and body weight change, male and female pup body weight and body weight change, feed consumption, feed utilization, organ weight, organ-to body weight ratio, gestation period, litter size, and pup percent survival data were evaluated using Bartlett’s test (p≤ 0.01), one-way analysis of variance (ANOVA) (p≤ 0.05), and Dunnett’s t-test (p≤ 0.05) to indicate statistical significance (MINITAB Statistical Software, State College, PA).

When the variances of the means were not considered by the Bartlett’s test (p≤ 0.01), the data were evaluated using a Kruskal-Wallis H-test (p≤ 0.05) followed by Mann-Whitney U-test (p≤ 0.05) (MINITAB Statistical Software, State College, PA).

The reproductive performance of the dams and the fertility and fecundity indices were evaluated in contingency tables, using a Chi-square test (p≤ 0.05). The total number of pups per litter (live and dead) and the total number of live pups per litter were evaluated using a linear regression model (p≤ 0.05).
Reproductive indices:
The following parameters were calculated to evaluate the effects on reproductive performance:

-Fertility index= (Number of pregnant females)(100)/(Number of cohabited pairs)
-Fecundity index= (Number of pregnant females)(100)/(Number of mated pairs with copulatory plug or sperm)
-Precoital interval= Date of Insemination-Date of first cohabitation
-Gestation duration= Date of Insemination-Date delivery begins
-Post-implantation loss= (Number of implantation sites-Number of pups in the litter)(100)/(Number of implantation sites)
-Pre-implantation loss= (Number of corpora lutea-Number of implantation sites)(100)/(Number of corpora lutea)

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
effects observed, treatment-related
Reproductive performance:
effects observed, treatment-related

Details on results (P0)

CLINICAL SIGNS: No mortality. Clinical signs were minimal to moderate and included: reductions in amount of feces for 16 high-dose and 2 mid-dose group rats for 1-6 days; minimally softened feces for 1 day in two high-dose group rats; dehydration in 1 rat each from the mid- and high-dose groups for 1-2 days; alopecia for 1-4 rats per dose group for 3-28 days; ocular or nasal porphyrin discharges in 1 rat per dose group for 1-12 days. One rat from the high-dose group had poor body condition (possibly related to malocclusion) on two days of the study. There were no other abnormalities observed.

BODY WEIGHTS: Mean body weights were 7% lower (p≤ 0.05) for female rats from the high-dose group on gestation day (GD) 20. Mean body weight gains were 27% and 48% lower (p≤ 0.05) for male and female rats, respectively, from the high-dose group on Day 7 of the pre-mating phase. No other changes in body weight or body weight gains were noted.

FEED CONSUMPTION AND FEED UTILIZATION: Mean feed consumption and feed utilization values were ~ 15% lower (p≤ 0.05) for male rats from the high-dose group on Day 7. Mean feed consumption values were 21% lower (p≤ 0.05) for female rats from the high-dose group on Day 7 of the pre-mating phase, 4% lower on GD 7, and 14% lower on postnatal day (PND) 4. There were no other differences in feed consumption or feed utilization patterns.

REPRODUCTIVE FUNCTION, SPERM MEASURES: Treatment-related reproductive effects observed in male rats included lower (p≤ 0.05) mean absolute and/or relative (to testis weight) testicular sperm counts for the high- and low-dose groups, and lower (p≤ 0.05) mean absolute epididymal sperm counts for all treated groups. The weight-adjusted epididymal sperm counts in the treated groups were also slightly lower but the difference was not statistically significant. There was no treatment-related effect on mean sperm motility. Group mean values for sperm motility were comparable between the study groups and ranged from 87% to 92%. Although lower than the control group, the epididymal sperm counts from the TXIB-treated groups were comparable to each other and were not reduced in a clear, dose-dependent manner. The apparent reduction appeared to be related to slightly higher control group values rather than a treatment-related effect. The reduction of the number of sperm per tissue and weight-adjusted testicular spermatid heads in the 15.0 mg/kg group occurred in the absence of a change in epididymal sperm counts. Therefore, the biological significance of the reduction in testicular sperm count was unclear.

REPRODUCTIVE PERFORMANCE AND LITTER DATA: There was a statistically significant reduction (~15%) in the mean number of implantation sites for dams from the high-dose group. Mean litter weights on PND 0 and 4 were decreased 19-20% and the number of live pups on PND 4 were 17% lower (p ≤0.05) for litters from the high-dose group. The mean number of pups dying between PND 0 and 4 was higher (p ≤0.05) for litters from the mid-dose group. All other examined parameters (reproductive performance; fertility index; fecundity index; precoital interval; gestation duration; percent survival; pre- and post-implantation loss; live and dead pups on postnatal day 0; percentage of male and female pups; and mean pup body weight and pup body weight change) were comparable among groups.

ORGAN WEIGHTS: All mean absolute, relative to body weight organ weights and terminal body weights for male rats were comparable among groups.

GROSS PATHOLOGY: Gross lesions observed at necropsy included minimal to minor alopecia of the forelimbs for 1-2 rats from the control, low- and high-dose groups, minor thymic hemorrhage for 1 control rat, and a cream colored nodule on the inner wall of the vagina with a mass on the outer surface of the vagina for 1 control rat. No other gross lesions were observed at necropsy.

HISTOPATHOLOGY: No treatment-related changes were detected during microscopic examination of the ovaries, right testis, epididymis, and gross lesions.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
276 mg/kg bw/day
Sex:
male
Basis for effect level:
other: At the highest dose level tested, there was a statistically significant decrease in the total number of implants. There was insufficient information in the study design to determine which sex was responsible for the effect.
Dose descriptor:
NOAEL
Effect level:
359 mg/kg bw/day
Sex:
female
Basis for effect level:
other: see 'Remark'

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Details on results (F1)


VIABILITY: The mean number of pups dying between Days 0 and 4 postpartum was statistically higher (p≤ 0.05) for litters from the mid-dose group. The mean number of live pups on Day 4 postpartum was 17% lower (p≤ 0.05) for litters from the high-dose group when compared with the control group.

CLINICAL SIGNS: Clinical abnormalities were observed for pups from all dose levels with similar frequencies of occurance between treated and control groups. These abnormalities included bruising, the pups appearing small (runts), and the pups having little or no milk in their stomachs. Additionally, pups were occasionally missing (presumably cannibalized) or found dead.

BODY WEIGHT: Mean litter weights on Days 0 and 4 postpartum were 19-20% lower (p≤ 0.05) for litters from the high-dose group when compared with control group.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Effects of Exposure to 2,2,4-Trimethyl-1,3-pentanediol diisobutyrate in Pregnant Female Rats

  2,2,4-Trimethyl-1,3-Pentanediol Diisobutyrate
Parameter (mg/g diet)
  0 1.5 4.5 15
Number bred 12 12 12 12
Number pregnant 12 11 12 12
Reproductive failure 0 1 0 0
Corpora Lutea of Pregnancy 16.6±2.5 16.6±2.7 16.8±5.8 13.9±2.9
Fertility Index

100.00%

91.70% 100.00% 100.00%
Fecundity Index 100.00% 91.70% 100.00% 100.00%
Precoital Interval 2.9±1.9 2.1±0.7 2.3±1.0 2.0±1.0
Total No. of Implants 15.2±0.9 15.1±1.9 14.7±2.3 12.8*±1.8
Pre-implantation loss 7.20% 7.80% 6.30% 6.40%
Post-implantation loss 3.30% 6.00% 6.60% 4.40%
Gestation Length 21.9±0.5 21.8±0.4 21.8±0.4 21.7±0.5
Percent Males on Lactation day 0 (%) 55.2 49 52.9 49.3
No. of Live Pups        
Day 0 14.5 14.1 13.5 12.2
         
Day 4 14.5 13.8 13 12.0*
# Pups Dying 0.0±0.0 0.1±0.3 0.5*±0.7 0.2±0.4
Days 0-4
Mean pup weights (g) ± SD        

 

 

 

 

Day 0

6.61±0.43

6.69±0.36

6.55±0.55

6.42±0.53

 

 

 

 

Day 4

9.97±0.67

9.98±0.54

10.05±1.05

9.7±1.00

Litter weight (g)

 

 

 

 

Day 0

95.58±8.97

94.44±15.97

87.72±14.05

77.6*±13.31

 

 

 

 

 

Day 4

144.07±12.15

137.00±17.19

129.01±16.43

115.02*±17.87

* Statistically different from control

Applicant's summary and conclusion

Conclusions:
Under conditions of this study, developmental or reproductive toxicity in male and female rats administered 2,2,4-trimethyl-1,3-pentanediol diisobutyrate in the diet at concentrations of 0 to 15 mg/g was limited to the high-dose group and was evidenced by a statistically significant decrease in the total number of implants, reduced litter weights on postnatal days 0 and 4, and increased mortality in the offspring on postnatal day 4. Treatment-related systemic effects in the parental generation were also primarily found in the high-dose group. The NOAEL for developmental and reproductive effects was 4.5 mg/g test material in the diet which resulted in doses of 276 mg/kg bw/day and 359 mg/kg bw/day in males and females, respectively.
Executive summary:

In a reproductive/developmental toxicity screening study, 2,2,4-trimethyl-1,3-pentanediol diisobutyrate was administered to 12 Sprague-Dawley rats/sex/group at dose levels of 0, 1.5, 4.5 and 15.0 mg/g ad libitum in the diet for up to 51 days. At the high-dose level, there was a statistically significant decrease in total number of implants, number of live pups on postnatal day 4, and litter weight on postnatal days 0 and 4. There were no statistically significant adverse effects on reproductive performance; fertility index; fecundity index; precoital interval; gestation duration; percent pup survival; pre- and post-implantation loss; live and dead pups on postnatal day 0; percentage of male and female pups; mean pup body weight and pup body weight change; or reproductive organ weights in the adults. Reductions in body weight and feed consumption values in the high-dose group adults were transient in nature and not considered toxicologically significant. Although minimal reductions in sperm counts were seen in the testes and/or epididymides of treated male rats, there were no treatment-related gross or microscopic lesions in any groups and no adverse effect on reproductive performance. The NOAEL for developmental or reproductive toxicity was 4.5 mg/g in the diet which was equivalent to 276 mg/kg bw/day for males and 359 mg/kg bw/day for females.