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Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
publication
Title:
Two-Generation Reproduction Study of Di-2-Ethylhexyl Terephthalate in Crl:CD rats
Author:
Faber WD, Deyo JA, Stump DG, Ruble K
Year:
2007
Bibliographic source:
Birth Defects Research (Part B) 80: 69-81

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) terephthalate
EC Number:
229-176-9
EC Name:
Bis(2-ethylhexyl) terephthalate
Cas Number:
6422-86-2
Molecular formula:
C24H38O4
IUPAC Name:
bis(2-ethylhexyl) terephthalate
Test material form:
liquid
Specific details on test material used for the study:
- Source: Eastman Chemical Company, Kingsport, TN
- Appearance: clear, colorless liquid
- Purity: > 97 %
- Stability: Stability under test conditions: confirmed (stability, homogeneity and verification of DEHT concentration in feed was confirmed by HPLC/UVD)
- Storage Conditions: room temperature

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Raleigh, NC, USA)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (F0) 52 days (42 days at arrival)
- Weight at study initiation: n.a.
- Housing: Until pairing, all F0 and F1 parental test animals were individually housed in clean wire-mesh cashes suspended above cage-board. During cohabitation, the animals were paired for mating in the home cage of the male. After evidence of mating, the males were again housed individually until the scheduled necropsy of the parental generations, and the females were transferred to plastic maternity cages with nesting material (Bed-O’Cobs; The Andersons, Industrial Products Division, Maumee, OH), The dams were housed in these cages until weaning on LD21. Animals were housed in accordance with the ‘‘Guide for the Care and Use of Laboratory Animals’’ (National Research Council, 1996).
- Diet (e.g. ad libitum): ad libitum, animals were fed PMI Nutrition International, Inc., Certified Rodent LabDiet 5002
- Water (e.g. ad libitum): ad libitum, municipal water was reverse-osmosis-treated (on-site) and delivered by an automatic watering system.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): No data
- Humidity (%): No data
- Air changes (per hr): No data
- Photoperiod (hrs dark/light): No data
As the study was conducted according to the OECD 416 testing guideline, it can be assumed that suitable conditions were met.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): every two weeks
- Storage temperature of food: room temperature
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days or until evidence of mating
- Proof of pregnancy: Mating pairs were examined daily for the presence of a copulatory plug or the presence of sperm in a vaginal smear. The day when evidence of mating was identified was termed day 0 of gestation (GD0).
- When evidence of mating did not occur within 14 days the female was placed in a maternity cage with no further opportunity for mating.
- After successful mating each pregnant female was caged (how): females were transferred to plastic maternity cages with nesting material (Bed-O’Cobs; The Andersons, Industrial Products Division, Maumee, OH) and were housed in these cages until weaning on lactation day (LD) 21.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability, homogeneity and verification of DEHT concentration in feed was confirmed by HPLC/UVD.
Duration of treatment / exposure:
- For at least 70 consecutive days before mating for the F0 and F1generations.
- Exposure for the F0 and F1 males continued throughout the mating period until euthanasia.
- Exposure for the F0 and F1 females continued throughout mating, gestation, and lactation until euthanasia
Frequency of treatment:
daily (ad libitum)
Details on study schedule:
- F1 parental animals were 13 weeks old when mating was started after selected from the F1 litters.
- Selection of parents from F1 generation: Offspring was weaned on LD 21 and those selected to continue on the study were provided diets containing the same level of DEHT as their parents.
- Age at mating of the mated animals in the study: F0: ca. 18 weeks, F1: 13 weeks
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg diet
Remarks:
Control group
Dose / conc.:
3 000 mg/kg diet
Remarks:
0.3% nominal in diet
Dose / conc.:
6 000 mg/kg diet
Remarks:
0.6% nominal in diet
Dose / conc.:
10 000 mg/kg diet
Remarks:
1% nominal in diet
No. of animals per sex per dose:
30
Control animals:
yes, plain diet

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for moribundity, appearance, behaviour, and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed physical examinations were recorded weekly for all F0 and F1 parental animals throughout the study period. Mated F0 and F1 females were also observed twice daily during the period of expected parturition and at parturition.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual F0 and F1 male body weights were recorded weekly throughout the study and just before necropsy. Individual F0 and F1 female body weights were recorded weekly until evidence of copulation was observed. Once evidence of mating was observed, female body weights were recorded on GD 0, 4, 7, 11, 14, 17, and 20 and on LD 1, 4, 7, 14, and 21.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: yes
Individual F0 and F1 male and female feed consumption was measured on a weekly basis except during the mating period. Female feed consumption was also recorded on GD 0, 4, 7, 11, 14, 17, and 20 and LD 1, 4, 7, 14, and 21.
Oestrous cyclicity (parental animals):
Vaginal smears were carried out daily for assessment of oestrous cyclicity, beginning 21 days before pairing. Vaginal smears were carried out on females also on the day of their euthanasia to determine the stage of oestrus.
Sperm parameters (parental animals):
Parameters examined in all male parental generations:
Spermatogenic endpoints were evaluated using the methods described by Nemec et al. (2004). Immediately after euthanasia the right epididymis of each F0 and F1 male was exposed, excised, and weighed. Sperm motility was assessed using the Hamilton-Thorne HTM-IVOS Version 12 computer-assisted sperm analysis (CASA) system. Sperm morphology was evaluated by light microscopy via a modification of the wet-mount evaluation technique (Linder et al., 1992). The left testis and cauda epididymis from all F0 and F1 males from all exposure groups were weighed, homo- genized, and a sample was evaluated for determination of homogenization-resistant spermatid count and sperm production rate (Blazak et al., 1985) using a DNA-specific fluorescent dye and the HTM-IVOS CASA system.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, 8 × F1 and F2 pups from each litter were selected randomly with equal gender distribution; excess pups were weighed, killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in all pups:
Number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, detailed physical examination on PND 1, 4, 7, 14 and 21. Balanopreputial separation and vaginal perforation were used to assess attainment of sexual maturation of the selected F1 pups (1/sex/litter).

GROSS EXAMINATION OF DEAD PUPS
Offspring that died between PND 0–4 were necropsied using a fresh dissection technique (Stuckhardt and Poppe, 1984). A detailed gross necropsy was carried out on any pup dying after PND 4 and before weaning.
Postmortem examinations (parental animals):
SACRIFICE
- All F0/F1 parental animals were sacrificed 6 to 10 days after weaning of litters. Female animals that experienced total litter loss were euthanized within 24 hours. F1 weanlings not selected for the next generation and F2 pups were euthanized on PND 21.

GROSS NECROPSY
- Gross necropsy was performed on all parental animals and weanlings, selected organs were weighed.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Selective histopathologic examination was carried out on 10 parental animals per gender in the control and high dose groups. In addition, uteri and vaginas from all F0, and F1, adult female animals in the control and high dose groups were examined histopathologically. Moreover, uteri and vaginas from all F0 and F1 adult female animals in the low- and mid-dose groups with uterine weights > 1 g were also examined.
The following organs were weighed after termination: Brain, liver, kidneys, spleen, seminal vesicles/coagulating gland, prostate, testis (right and left), epididymis (right and left), cauda epididymis (right and left), uterus, ovaries, thymus gland, adrenal glands, pituitary gland. No detailed overview of tissues prepared for microscopic examination is given in the publication.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed on PND 21.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: A complete necropsy was conducted on all weanlings. In the publication no further information was provided.
Statistics:
All analyses were two-tailed using a minimum significance level of 5 %. The following statistical analyses were carried out: Chi-squared test with Yates’ correction factor (Hollander and Wolfe, 1999) for parental mating and fertility indices; one-way analysis of variance (ANOVA) with Dunnett’s test (Dunnett, 1964) for body weights (adult and pup from PND 4–21), feed consumption, organ weights, sperm numbers, pre-coital intervals, live litter size, and acquisition of developmental landmarks; Kruskal-Wallis test with Mann-Whitney U-test (Kruskal and Wallis, 1952) for sperm motility, sperm morphology, pup gender ratio, and postnatal survival; Fisher’s exact test (Steel and Torrie, 1980) for histopathologic data; and analysis of covariance (with litter size as the covariate) and Student’s t-test (SAS Institute, Inc., 1997) for offspring weights before standardization of litters on PND 4.
Reproductive indices:
Oestrous cycle length, precoital interval, mating index, fertility index, gestation length
Offspring viability indices:
Total pups/litter, live pups/litter, gender ratio and pup survival

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were detected in males and females in either generation that survived to scheduled euthanasia.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Lethality was observed in F0 and F1 dams consuming the 1.0 % diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3 % or 0.6 % DEHT.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean weekly body weight gain in the F0 males in the 1.0 % group was reduced (15–25 %) during weeks 3–4, 4–5, and 6–7, resulting in a slightly reduced (5 %) mean body weight at termination in this group. No other differences in body weight or body weight gain were observed in the remaining groups of F0 males and females during the pre-mating period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption for the F0 male and female rats was unaffected by DEHT exposurethroughout the entire generation (males) or during the pre-mating period (females).
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No DEHT-related macroscopic or microscopic findings were observed for the parental animals in either generation.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cycle of the F0 were unaffected by test diet administration of DEHT at all concentrations. Please see Table 2 in box "Any other information on results incl. tables".
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic assessment parameters (e.g. motility, morphology) in the F0 and F1 males were unaffected by DEHT exposure. Please see Table 3 in box "Any other information on results incl. tables".
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive performance parameters (e.g. mating and fertility parameters, gestation lengths) for the F0 were unaffected by test diet administration of DEHT at all concentrations. Please see Table 2 in box "Any other information on results incl. tables".

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
10 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse signs of reproductive toxicity were observed
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
3 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity (P0)

Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 mg/kg diet
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were detected in males and females in either generation that survived to scheduled euthanasia.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
Lethality was observed in F0 and F1 dams consuming the 1.0 % diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3 % or 0.6 % DEHT.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced mean body weight gain in the 0.6 % and 1.0 % F1 males groups, along with lower mean birth weights and decreased growth before weaning from the F0 maternal animals, resulted in reduced mean body weights throughout the generation for the F1 males in the 0.6 % and 1.0 % group. Similar to the F0 females, F1 female body weight gain after weaning in the 0.6 % and 1.0 % groups was similar to the control group (data not shown). However, as a result of reduced mean body weights during the pre-weaning period in the 0.6 % and 1.0 % F1 female group, pre-mating body weights in these groups were decreased relative to the control group, although only the 1.0 % group was significant statistically. No test article-related effects on mean weekly body weights were observed in the 0.3 % group F1 males and female groups.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Feed consumption for F1 female rats was unaffected by DEHT exposure throughout the entire generation (males) or during the pre-mating period (females). Correlating with the reduced mean body weights, feed consumption in the 0.6 % and 1.0 % F1 males was slightly reduced (10 %) during the first week after weaning (0.6 % group) or throughout the generation (1.0 % group); the differences from the control group were often statistically significant.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Liver weight relative to final body weight was increased in both the F0 and F1 females in the 0.6 % and 1.0 % groups (p < 0.05). The increases in liver weight were attributed to DEHT exposure. These increases in liver weights were not unexpected, as this is a characteristic finding in animals exposed to high dose levels of DEHT. High dose levels of 2-ethylhexanol (a metabolite of DEHT) has been shown to cause an acute phase response and hypertrophy (Astill et al., 1996) in the liver when administered by oral gavage. Several statistically significant decreases were observed in mean absolute organ weights in the F1 adults. In most cases, however, these changes disappeared when compared relative to the body weights of the animals suggesting the differences were due to the decreased body weights. In addition, no correlative macroscopic or microscopic findings were observed in these tissues. Therefore the differences from the control group were not considered to be biologically relevant or adverse.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No DEHT-related macroscopic or microscopic findings were observed for the parental animals in either generation.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No DEHT-related macroscopic or microscopic findings were observed for the parental animals in either generation.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cycle of the F0 were unaffected by test diet administration of DEHT at all concentrations. Please see Table 2 in box "Any other information on results incl. tables".
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Spermatogenic assessment parameters (e.g. motility, morphology) in the F0 and F1 males were unaffected by DEHT exposure. Please see Table 3 in box "Any other information on results incl. tables".
Reproductive performance:
no effects observed
Description (incidence and severity):
Reproductive performance parameters (e.g. mating and fertility parameters, gestation lengths) for the F1 were unaffected by test diet administration of DEHT at all concentrations. Ovarian follicle counts for the F1 females in the high-exposure group were similar to the control values. Please see Table 2 in box "Any other information on results incl. tables".

Effect levels (P1)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive toxicity
Effect level:
10 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse signs of reproductive toxicity were observed.
Key result
Dose descriptor:
NOAEL
Remarks:
parental toxicity
Effect level:
3 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity (P1)

Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 mg/kg diet
System:
hepatobiliary
Organ:
liver
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were detected in males and females in either generation.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality were observed.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
F1 male and female pup weights in the 0.3, 0.6, and 1.0 % groups were significantly reduced on PND 1 and 4. The mean pup weights in the F1 1.0 % group start to fall below the historic control group values by PND 7, with further reductions throughout the lactation period (coincidentally with decreases in feed consumption by the F1 1.0 % group dams). Despite the similarities with historic control group values, the replication of reduced mean pup body weights from PND 14–21 in the 0.6 % and 1.0 % group from both the F1 and F2 generation pups were considered related to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Description (incidence and severity):
In the 1.0% F1 male animals a 5 % increase in age of balanopreputial separation was recorded. It is important to note that the mean day of attainment of balanopreputial separation in the 1.0 % F1 males was equal to the historic control value for this endpoint in the performing laboratory. In addition, There was no additional evidence of effects on male sexual development in the F1 male animals as all other measures of sexual function (fertility, sperm parameters, testicular weight, and histology) were unaffected. Therefore, the slight increase in age of balanopreputial separation in the 1.0 % group was not considered adverse. Thus, no adverse effects on sexual maturation were observed (balanopreputial separation; vaginal patency).
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although several changes in the PND 21 absolute organ weights from the 0.6 % group were observed in the F1 and F2 pups, these changes disappeared when they were adjusted for the decreased body weights of these animals (data not shown). Changes in mean F1 and F2 male and female pup organ weights on PND 21 that were unrelated to decreased body weights included a reduced (13 %) mean relative spleen weight in the F1 male 1.0 % group and the F2 male (8 %) and female (11 %) 1.0 % groups, and a reduced (12 %) mean relative thymus weight in the F2 female 1.0 % group. In addition, mean relative brain weights were increased for both sexes in the F1 (25 %) and F2 (23–25 %) 1.0 % group and the F1 0.6 % female group (12 %). Increases in relative brain weights in the F1 male and female animals from the 0.6 % and 1.0 % dietary groups were considered to be related to the decreased terminal body weights of these animals rather than a direct effect of DEHT exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings attributable to parental exposure to DEHT were noted at the scheduled necropsy of F1 and F2 pups euthanized on PND 21.
Histopathological findings:
not specified
Other effects:
not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
3 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity (F1)

Critical effects observed:
yes
Lowest effective dose / conc.:
10 000 mg/kg diet
Organ:
spleen
thymus
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were detected in males and females in either generation that survived to scheduled euthanasia.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In the F2 generation, the mean pup body weights in the 0.3 % and 0.6 % groups for PND 1 and 4 were comparable to concurrent control values. Although the PND 1 and PND 4 mean pup weights in the F2 1.0 % group were reduced significantly when compared to the concurrent control value, they were slight (3–4 %) and equivalent to historic control values. Thus these changes were considered of no biological relevance.
The mean pup weights in both the F1 and F2 1.0 % groups start to fall below the historic control group values by PND 7, with further reductions throughout the lactation period (coincidentally with decreases in feed consumption by the F1 and F2 1.0 % group dams). On PND 14 and 21, the mean pup weights from the F2 0.6 % group were reduced significantly when compared to the concurrent control values but were higher than the corresponding historic control values. Mean pup body weights for the F2 0.3 % group were comparable to concurrent control and historic control values at all time points. Despite the similarities with historic control group values, the replication of reduced mean pup body weights from PND 14–21 in the 0.6 % and 1.0 % group from both the F1 and F2 generation pups were considered related to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
not examined
Nipple retention in male pups:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Although several changes in the PND 21 absolute organ weights from the 0.6 % group were observed in the F1 and F2 pups, these changes disappeared when they were adjusted for the decreased body weights of these animals (data not shown). Changes in mean F1 and F2 male and female pup organ weights on PND 21 that were unrelated to decreased body weights included a reduced (13 %) mean relative spleen weight in the F1 male 1.0 % group and the F2 male (8 %) and female (11 %) 1.0 % groups, and a reduced (12 %) mean relative thymus weight in the F2 female 1.0 % group. In addition, mean relative brain weights were increased for both sexes in the F1 (25 %) and F2 (23–25 %) 1.0 % group and the F1 0.6 % female group (12 %). Increases in relative brain weights in the F1 male and female animals from the 0.6 % and 1.0 % dietary groups were considered to be related to the decreased terminal body weights of these animals rather than a direct effect of DEHT exposure.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings attributable to parental exposure to DEHT were noted at the scheduled necropsy of F1 and F2 pups euthanized on PND 21.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
3 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios

Target system / organ toxicity (F2)

Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Analysis of diet

Analysis of diets containing DEHT indicated that the formulations were stable, homogenous, and within 10% of the target concentration.

Mean calculated Compound Consumption:

Table 1: Mean calculated compound consumption in mg/kg bw/day
  Males Females
Nominal Dietary Level Before Breeding After Breeding Before Breeding Gestation Lactation After Weaning
F0 generation
0.3% 182 133 223 184 478 207
0.6% 367 265 449 372 940 419
1.0% 614 447 783 595 1349 745
F1 generation
0.3% 256 159 275 206 516 227
0.6% 523 320 573 423 1036 486
1.0% 893 552 1021 697 1549 868

Reproductive parameters:

Table 2: Reproductive Parameters and Litter data for F1 Generation
Parameter 0 0.3 % 0.6 % 1.0 %
Mating pairs (n) 30 30 30 30
Estrous cycle length (days) 4.9 +/- 1.98 5.2 ± 1.24 4.8 ± 1.39 5.6 ± 2.90
Precoital interval (days) 3.7 ± 2.36 3.0 ± 1.38 3.1 ± 1.28 2.6 ± 1.62
Mating index (%) 93.3 100 100 93.3
Fertility index (%) 80.0 80.0 93.3 93.3
Gestation length (days) 21.6 ± 0.49 22.0 ± 0.32* 21.9 ± 0.2 21.9 ± 0.51
Total pups/litter (n) 14.3 ± 3.29 14.3 ± 3.25 14.5 ± 2.19 14.2 ± 2.13
Live pups/litter (n) 13.9 ± 3.18 14.0 ± 3.16 14.2 ± 2.02 13.7 ± 2.83
Gender ratio (% per litter) 50.3 ± 13.73 54.2 ± 13.96 51.2 ± 11.88 46.0 ± 14.82
Pup survival (% per litter)      
PND 0 97.6 ± 4.23 98.2 ± 3.86 98.0 ± 4.11 96.5 ± 13.62
PND 0-1 99.5 ± 1.76 99.5 ± 1.78 99.5 ± 1.89 98.1 ± 4.61
PND 0-4 (precull) 96.3 ± 4.93 97.1 ± 5.24 97.0 ± 4.32 94.7 ± 14.40
PND 4 (precull) - 21 99.5 ± 2.55 99.0 ± 3.53 100 ± 0.0 100 ± 0.0
*= p< 0.05

Spermatogenic assessments:

Table 3: Spermatogenic assessments
Parameter 0 0.3 % 0.6 % 1.0 %
F0 males
rats examined 29 30 29 30

Sperm concentration

(× 106/g)

     
left testis 105.4 ± 40.75 102.7 ± 27.92 111.6 ± 28.25 104.4 ± 25.58
left epididymis 454.6 ± 126.55 448.9 ± 132.08 460.0 ± 104.61 459.3 ± 98.61
Motility (%) 91.1 ± 3.52 90.5 ± 3.57 89.1 ± 4.86 89.3 ± 4.74
Morphology (% normal) 99.8 ± 0.43 99.8 ± 0.45 99.8 ± 0.34 99.8 ± 0.31
F1 males
rats examined 30 30 30 29

Sperm concentration

(× 106/g)

     
left testis 83.8 +/- 19.27 81.4 +/- 17.19 81.6 +/- 16.15 87.9 +/- 27.33
left epididymis 412.9 +/- 90.02 413.7 +/- 91.93 416.1 +/- 86.18 427.1 +/- 109.51
Motility (%) 85.3 +/- 12.99 86.0 +/- 7.20 81.7 +/- 13.94 85.7 +/- 9.37
Morphology (% normal) 99.9 +/- 0.23 99.5 +/- 0.84 99.6 +/- 0.93 99.7 +/- 0.34

Applicant's summary and conclusion

Conclusions:
In a two-generation reproductive toxicity study conducted according to OECD 416, rats were exposed via diet to 10000, 6000 and 3000 mg/kg diet. Based on the results, the NOAEL for systemic toxicity in both growing and adult animals is 3000 mg/kg diet. The NOAEL for reproductive toxicity is 10000 mg/kg diet.
Executive summary:

In a two-generation reproductive toxicity study conducted according to OECD 416 groups of male and female Crl:CD rats (30/sex/dose) were exposed via diet to 10000, 6000 and 3000 mg/kg diet of di-2-etyhlhexyl terephthalate (DEHT). Parental animals were exposed for at least 70 consecutive days before mating for the F1 and F2 generations. Exposure of the males continued throughout the mating period until euthanasia. Exposure of the females continued throughout mating, gestation and lactation. F1 and F2 pups were weaned on postnatal day (PND) 21.

Exposure to DEHT did not affect clinical observations. However, mortality was observed in P0 and P1 dams consuming 10000 mg/kg diet during the post-weaning period. No treatment-related mortality occurred in any of the male groups exposed to DEHT or in the female groups exposed to 0.3 % or 0.6 % DEHT. Male rats consuming the 1.0 % in both parental generations gained weight more slowly than the controls. There were no indications of adverse effects on reproductive performance in either generation. Male and female mating and fertility indices, pre-coital intervals, spermatogenic endpoints, reproductive organ weights, lengths of oestrous cycle and gestation, live litter size, developmental landmarks, and postnatal survival were similar in all exposure groups. Additionally, ovarian follicle counts for the F1 females in the high-exposure group were similar to the control values. No adverse exposure-related macroscopic pathology was noted at any exposure level in either generation. Increases in liver weights were found in the male and female animals exposed to 6000 or 10000 mg/kg diet. Because there were no accompanying histopathologic changes, this effect was not considered adverse. Significant decreases in feed consumption in the female animals from the groups consuming 10000 mg/kg diet during lactation accompanied reduced postnatal pup body weights and rate of weight gain. Reductions in pup body weights later in lactation may also have been due to direct consumption of the treated feed by the pups or taste aversion to the same. Reduced relative spleen weight was found in male weanling pups from the high dose group in both generations and reduced relative spleen and thymus weights were found in female pups from the high dose group in the F2 generation at necropsy on PND 21. Therefore, for parental and pup systemic toxicity, 3000 mg/kg diet was considered as the NOAEL. As no adverse effects were seen for reproductive toxicity, 10000 mg/kg diet was considered the NOAEL for both generations.

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