Bis[C5-(linear and branched)-alkyl] benzene-1,4-dicarboxylate

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 Apr 2021 - 09 Aug 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

June 2018

GLP compliance:

yes

Justification for study design:

The oral gavage route of exposure was selected because this is the most appropriate route of administration for substances to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (Chapter R.7a, Section R.7.6.2.3.2) and it is an possible route of human exposure.
The dose levels have been selected based on available toxicological data and the results of a preliminary reproduction/developmental toxicity study in rats (2019-05286-ENR) with oral (gavage) administration.
The purpose of this preliminary study was to provide information on systemic toxicity of Elatur® DPT/Di-(iso)-pentyl terephthalate in pregnant and lactating females, young and adult offspring following exposure of the F0 females from Day 0 of gestation until weaning of F1 pups (Day 20 post partum). Each group comprised 10 female F0 rats. Selected F1 pups (10 male and 10 female per group) were dosed starting from Day 21 of age until Day 42 of age and sacrificed on Day 43. Dose-levels for F0 females and F1-generation were 0, 500 and 1000 mg/kg/day. The vehicle used was corn oil and the dose volume was 2 mL/kg body weight. Results of toxicological effects were limited to salivation and some tendencies to
reduced maternal body weight (gain) in the high dose group. This, however, was limited to only some stages of pregnancy and/or lactation. There were no dose related findings recorded at macroscopic observations. Litter data in terms of litter weight and mean pup weight, from the birth until weaning in groups receiving 500 and 1000 mg/kg/day did not show effects clearly connected to the test item. Based on the results of this study, it was concluded that the high dose level to use in the subsequent study could be 1000 mg/kg/day, both for parental and selected F1-generation.
Hence, dose level of 100, 300 1000 mg/kg bw/day were selected for this study.

Test material

Test material information

Constituent 1

Reference substance name:

Bis[C5-(linear and branched)-alkyl] benzene-1,4-dicarboxylate

EC Number:

940-272-6

Cas Number:

2097734-13-7

Molecular formula:

C18H26O4

IUPAC Name:

Bis[C5-(linear and branched)-alkyl] benzene-1,4-dicarboxylate

Test material form:

liquid

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) Sprague Dawley

Details on species / strain selection:

The Sprague Dawley rat was chosen as the animal model for this study as it is an accepted rodent species for nonclinical toxicity testing by regulatory agencies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Margate, Kent, UK
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: males were between 49 and 55 days old and females were between 56 and 62 days old at the initiation of dosing
- Weight at study initiation: (P) Males: 212 - 320 g; Females: 186 - 253 g
- Fasting period before study: no
- Housing: Group housed (up to 4 animals of the same sex and same dosing group together).
A few days prior to mating, F0 and Cohort 1B males were transferred to individual cages with solid bottoms. F0 and Cohort 1B females were transferred to these cages for mating.
Mated F0 and Cohort 1B females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. F0 and Cohort 1B females with litters were retained in this type of cage until termination.
On a suitable day after completion of mating, the F0 and Cohort 1B males were re-housed with their original cage mates.

- Diet (e.g. ad libitum): Ad libitum, except during designated procedures
- Water (e.g. ad libitum): Freely available to each animal from water bottles (except during designated procedures).
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target 19 to 23°C. Actual 19 to 25°C.
- Humidity (%): Target 40 to 70%. Actual 29 to 72%. There were occasions where the target environmental conditions for temperature and humidity were not maintained. The majority of these occasions were transient, however on one occasion the conditions for humidity were out of specification for a time period that were considered a reportable incident, down to a minimum of 29%. The health of the animals was unaffected on any occasion, therefore, these excursions were considered not to impact the outcome or integrity of the study.
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)
IN-LIFE DATES: From: 21.04.2021 To: 25.01.2022

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): solubilty of test substance
- Concentration in vehicle: 500, 150, 50 mg/ml
- Amount of vehicle (if gavage): 2 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: During the evening (after 5 pm), females were housed with their allocated co-group male partner. The animals were paired in ascending numerical order. The pairing period for each pair of animals was a maximum of 14 nights. If evidence of mating was not observed by the end of the pairing period, the female was separated from the male during the morning following the last night of pairing and treated as if mating had occurred during that night. Procedures for that female continued as if it had mated on the last night of pairing. For each female the time taken to show a positive mating sign and the number of failed opportunities to mate (estrouses passed without a sign of mating) was evaluated.
- Proof of pregnancy: vaginal plug referred to as day GD0 of pregnancy
- After successful mating each pregnant female was caged: Mated F0 and Cohort 1B females were transferred to individual solid bottomed cages. White paper tissue was supplied as nesting material from Gestation Day (GD) 20. F0 and Cohort 1B females with litters were retained in this type of cage until termination.

Duration of treatment / exposure:

F0 Males: 10 weeks prior to mating and continuing throughout and after mating until the day before termination.
F0 Females: 10 weeks prior to mating and continuing throughout mating and gestation until at least Lactation Day (LD) 21.
F1 Surplus PND 52 Pups: From PND 21 until the day before necropsy (at least PND 52).
Cohort 1A: From PND 21 until the day before necropsy (at least PND 90).
Cohort 1B: From PND 21 until the day before necropsy (at least LD 21).

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day

Remarks:

mid dose

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

high dose

No. of animals per sex per dose:

F0: 25
F1: Cohort 1A: 20 Cohort 1B: 25 F1 PND 52 Surplus Pups: 15 F1 Unselected PND 22-24 Pups: 10
F2: PND 22-24 Pups: 10

Control animals:

yes, concurrent vehicle

Details on study design:

administration volume: 2 ml/kg bw/day

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly;

BODY WEIGHT: Yes
- Time schedule for examinations:
F0:
Males: weekly beginning Week -1.
Females: weekly beginning Week -1 until pairing for mating and then on GD 0, 7, 14 and 20 and LD 1, 4, 7, 14 and 21. A body weight was also taken on LD 0 if necessary for dosing purposes only and not reported but maintained in the study data.
Pups were weighed individually on PND 1, 4, 7, 14 and 21

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes Males: weekly from Week 1 until the males were separated for pairing for mating. Then weekly after mating and re housing.
Females: weekly beginning Week -1 until pairing for mating and then on GD 0-7, 7-14 and 14-20, and LD 1-7, 7-14 and 14-21

WATER CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: Regular basis throughout the study. Monitored by visual inspection of the water bottles. No inter-group differences were noted.

MILK ANALYSIS: Milk samples were taken from 3 (control) or 6 (test item groups) randomly selected females between LD 10-13.
The milk samples were transferred to the analytical laboratory at the Test Facility.

Oestrous cyclicity (parental animals):

From 2 weeks prior to pairing until day of detection of a copulatory plug in situ and/or of sperm in the lavage. Until either mating was detected or the end of the mating period. Also, on the morning of necropsy
Vaginal lavages were taken early each morning and the stage of estrous observed was recorded.

Sperm parameters (parental animals):

Parameters examined in P and F1 male parental generations:
testis weight, epididymis weight, sperm count, sperm motility, sperm morphology, spermatid count

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); Any additional pups were selected at random for use in blood-sample or necropsy procedures. Where 5 males and 5 females were not available on PND 4, additional pups of the opposite sex were used to ensure there was a total of 10 pups. Where the total number of pups in the litter on PND 4 was 10 or fewer, no adjustment was performed.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, sexual maturation (females: daily examination for vaginal opening; pup weight on the day of vaginal opening; males: daily examination for balano preputial separation; pup weight on the day of balano preputial separation);

GROSS EXAMINATION OF DEAD PUPS: yes
Where practicable, any pups that were found dead or were killed during the postnatal period were sexed and appropriately examined as above. Any externally abnormal decedent pup was preserved; externally normal pups were discarded.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: cohort was not included in this study

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: cohort was not included in this study

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after the majority of F0 females
- Maternal animals: All surviving animals LD 22 - 24. . Any female that failed to litter was necropsied on the assumed GD 24. Any female that had a total litter loss was necropsied at a suitable time prior to LD 22-24.

NECROPSY
Complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues. The reproductive tracts of all F0 females were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female. For F0 females necropsied on GD 24, as they failed to produce a litter and the uteri of all non-pregnant females were fixed in buffered formalin and stained and examined for implantation sites. On GD 24, if a female was found to be pregnant, the number and type of any implantation sites were recorded and the total number of corpora lutea graviditatis was recorded also.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues according to OECD Testguideline 443 (2018) were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- F1 animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:
“Surplus” pups All On PND 52 Gross necropsy
All F1 and F2 unselected pups at PND 4 Gross Necropsy; Tissue collection only if abnormal tissue identified
All F1 unselected pups and all F2 pups at PND 22-24 Tissue collection and organ weights from 10 pups/sex/group only according to OECD Testguideline 443 (2018). All other pups had gross necropsy only performed.
Cohort 1B animals were subjected to a complete necropsy examination, which included evaluation of the carcass and musculoskeletal system; all external surfaces and orifices; cranial cavity and external surfaces of the brain; and thoracic, abdominal, and pelvic cavities with their associated organs and tissues.
The reproductive tracts of all Cohort 1B animals were examined for signs of implantation, the number of any implantation sites being recorded. The total number of corpora lutea graviditatis was recorded for each female.
GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissues according to OECD Testguideline 443 (2018) were prepared for microscopic examination and weighed, respectively.

Statistics:

Female Mating Index
Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)/ Number of Females Paired

Female Fertility Index
Number of Pregnant Females/Number of Females with Evidence of Mating (or no confirmed mating date and pregnant)

Female Pregnancy Index
Number of Pregnant Females/Number of Females Paired

Male Mating Index
Number of Males with Evidence of Mating (or female partner confirmed pregnant)/ Number of Males Paired

Male Fertility Index
Number of Males Impregnating a Female/Number of Males with Evidence of Mating (or female partner confirmed pregnant)

Male Pregnancy Index
Number of Males Impregnating a Female/Number of Males Paired

Reproductive indices:

Gestation Length: The gestation length was calculated from GD 0 to the day the first pup was observed.

Gestation Index: Percentage of pregnancies that resulted in birth of live litters:
(Number of Animals with Live Offspring/Number of Animals Pregnant) x 100

Live Birth Index Percentage of pups born alive:
(Number of Live Newborn Pups/Number of Newborn Pups) x 100

Sex Ratio (% males) Percentage of male pups per litter:
(Number of Live Male Pups/Total Number of Live Pups) x 100

Offspring viability indices:

Viability Index: Percentage of pups born that survived 4 days postpartum:
(Number of Live Pups on Day 4 Postpartum/Number of Live Newborn Pups) x 100
 
Lactation Index Percentage of pups that survived 21 days postpartum:
(Number of Live Pups on Day 21 Postpartum/Number of Live Pups on Day 4 (post culling) Postpartum) x 100

Post-Implantation Loss/Litter:
(Number of Implants – Total Newborn Pups)/Number of Implants x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a higher incidence of ploughing and salivation recorded in both F0 and F1 animals at all dose levels but particularly at 300 or 1000 mg/kg/day. These observations were recorded immediately to 1-hour post dose, they were dose-related therefore, were considered to be most likely a result of the taste and formulation of the test item thus, considered not to be toxic.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were six unscheduled deaths in F0 animals during the course of this study. None of these deaths were test item-related, due to no test-item histological findings.

At 1000 mg/kg/day, animal 4503F was euthanised on Study Day 99, the clinical observations noted were decreased respiratory rate, erect fur, both eyes partly closed, brown fur staining on the dorsal cervical region, thin, hyperreactive and subdued, this resulted in lack of maternal care and all pups dying. There were no gross necropsy observations and all microscopic findings were considered incidental. The cause of this animal death was undetermined. Animal 4511F and 4514F were euthanised on Study Day 98 and 97, respectively, all their pups had died due to cannibalisation. Animal 4511F was noted to have neonatal offspring material in the stomach and a minimal decrease in cortical lymphoid cellularity was noted microscopically in the thymus. All other findings were considered incidental. Animal 4514F, mottled discoloration was noted grossly in the lungs for which there was no microscopic correlate. A minimal decrease in cortical lymphoid cellularity was noted microscopically in the thymus. All remaining microscopic findings were considered incidental.

At 100 mg/kg/day Animal 2007M was found dead on Study Day 69, there were no clinical observations associated with the animal’s death. Gross necropsy findings indicated dark red foci in the thymus which correlated microscopically with mild agonal haemorrhage. The remaining microscopic findings were considered incidental, of the nature commonly observed in this strain and age of rat. The cause of this animal death was undetermined. Animal 2023M was euthanised on Study Day 95 on veterinary advice due to limited use of the left hindlimb and an abnormal gait, 3 days prior to necropsy the animal was treated with Meloxicam, there was no gross necropsy findings. Animal 2511F was euthanised on Study Day 98, as all pups had died.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall, administration of the test item at 1000 mg/kg/day was associated with lower body weights in males and females.
F0 Animals
In the F0 generation males dosed at 1000 mg/kg/day, had slightly lower body weight gains (Day 22 – Day 71 and Day 78 – Day 113 of dosing) with sporadic statistical significance. This resulted in slightly lower mean bodyweights on Day 71 (-3.7%, when compared with controls) and on Day 113 (-5.2%, when compared with controls).
In the F0 generation females dosed at 1000 mg/kg/day, had lower bodyweights during premating period of up to 4.7%, when compared with controls, on Day 71 of dosing. During gestation these animals had overall statistically significant lower body weight gain between GD 14 and GD 20 (mean bodyweight gain 70.1 g) when compared with controls (mean bodyweight gain 88.1 g) over the same time. This changed during lactation where the females had higher body weight gains (LD 4 - LD 7 mean bodyweight gain 15.4 g and LD 7- LD 14 main bodyweight gain 17.3 g), when compared with control during the same period (LD 4 - LD 7 mean bodyweight gain 7.8g and LD 4 - LD 14 mean bodyweight gain 13.8g). However, their absolute body weight remained slightly lower (-2%) on LD 21 (356.8 g) when compared with controls, (364.5 g).
There was no effect on body weight and body weight gain in males and females, dosed at 100 and 300 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F0 Animals
In F0 generation, during premating period females dosed at 1000 mg/kg/day had a lower mean food consumption, which was statistically significant (on Days 15 – 22 and Days 64 – 71), however, the average difference did not exceed 10%, when compared with controls.
Similarly, during gestation and lactation period, F0 females dosed at 1000 mg/kg/day had lower food consumption, (the mean difference was up to 13.32% and 8.6% lower, respectively) when compared with controls.
There were no test item related effects on food consumption of F0 females dosed at 100 or 300 mg/kg/day and F0 males dosed at levels of 100, 300 or 1000 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on hematology parameters at dose levels of 100, 300 or 1000 mg/kg/day. Differences in some haematology parameters attained statistical significance, however there was either no dose-related trend or were minor and of insufficient magnitude to be considered toxicologically relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on clinical chemistry parameters at dose levels of 100, 300 or 1000 mg/kg/day.
Differences in some cases attained statistical significance, however they were minor and of insufficient magnitude to be considered toxicologically relevant.

Endocrine findings:

no effects observed

Description (incidence and severity):

There were no test item related effects on TSH and T4 at dose levels of 100, 300 or 1000 mg/kg/day.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 or 1000 mg/kg/day there were statistically significant lower urine pH levels, in F0 males (6.00 or 6.25, respectively; control 8.40) and females (7.45 or 6.10, respectively; control 8.60) and in F1 Cohort 1A males (6.65 or 6.05, respectively; control 8.50) and females (6.80 or 6.30, respectively; control 8.11), when compared with controls.
Additionally, at 1000 mg/kg/day there were x 0.98-fold lower specific gravity values in F0 females. This finding was not reproducible in the next generation.
There were no test item-related effects on urinalysis parameters at dose levels of 100 mg/kg/day.

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related microscopic findings.
A cystic adenoma was evident microscopically in the mammary gland of 2518F dosed at 100 mg/kg/day, correlating with the inguinal skin mass noted grossly. This benign mammary gland tumour can occur spontaneously in Sprague-Dawley rats (Chandra 1992) and given that only a single animal dosed at 100 mg/kg/day was affected this was considered not to be test item-related.
Decreased cortical lymphoid cellularity was noted in the thymus of 3/22 females dosed at 1000 mg/kg/day (similar to that seen in decedents F4511 and F4514). However, the minimal nature and low incidence of this finding which only affected females was considered insufficient evidence of a DPT-related effect.
Other microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be DPT-related.

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Palpable mass:
From study day 57 to 119 (day of necropsy), there was a palpable mass recorded in F0 animal 2518F dosed at 100 mg/kg/day, in the right inguinal region, which was noted to be firm and blue in colouration (blue colouration seen from Day 71-119). At necropsy it was noted to be irregular shaped, mottled cross section lobular which oozed thick yellow fluid. This a benign cystic mammary gland adenoma and was considered incidental and not to be test item- related.

Milk analysis:
No residues of DPT were found in the untreated sample used for fortification of the QC sample at or above the LOQ 100 ng/ml. A residue greater than the LOQ (100 ng/ml) was found in untreated sample 1510.
Overall mean recovery efficiencies for DPT in rat milk were within the required range of 70 – 120% and the relative standard deviation was ≤20% at all fortification levels thus demonstrating sufficient accuracy and precision of the method used.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on females estrous cycles at dose levels of 100, 300 or 1000 mg/kg/day for F0 and F1 generation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item related effects in the sperm straight line velocity, the percentage motile sperm or percentage of progressive motile sperm at dose levels of 100, 300 or 1000 mg/kg/day.
There were no test item related effects on sperm morphology, sperm counts, or spermatid counts at dose levels of 100, 300 or 1000 mg/kg/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item-related effects on reproductive performance at dose levels of 100, 300 or 1000 mg/kg/day for F0 and F1 generation.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:

no

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no test item-related effects on females estrous cycles at dose levels of 100, 300 or 1000 mg/kg/day for F0 and F1 generation.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no test item related effects in the sperm straight line velocity, the percentage motile sperm or percentage of progressive motile sperm at dose levels of 100, 300 or 1000 mg/kg/day.
There were no test item related effects on sperm morphology, sperm counts, or spermatid counts at dose levels of 100, 300 or 1000 mg/kg/day.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no test item-related effects on reproductive performance at dose levels of 100, 300 or 1000 mg/kg/day for F0 and F1 generation.

Details on results (P1)

Moreover, there were no test item-related effects on the ovarian follicle count.

Effect levels (P1)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P1)

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There was a higher incidence of ploughing and salivation recorded in both F0 and F1 animals at all dose levels but particularly at 300 or 1000 mg/kg/day. These observations were recorded immediately to 1-hour post dose, they were dose-related therefore, were considered to be most likely a result of the taste and formulation of the test item thus, considered not to be toxic.
Furthermore, in the F1 Cohort 1B males and females, were noted to have ungroomed fur. In males this effected 12%, 32% and 28 % of animals dosed at 100, 300 or 1000 mg/kg/day, respectively. In premating females, this was noted in 12% or 24% of animals, dosed at 300 or 1000 mg/kg/day, respectively and during gestation and lactation it was 13% or 30% of females dosed at 300 or 1000 mg/kg/day, respectively. This a was dose-related trend, and this observation was only noted in this cohort, therefore was considered not adverse.
All other clinical observations were considered to be normal background findings commonly observed in this species.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

There were no test item-related effects on litter survival, viability index or lactation index at dose levels of 100, 300 or 1000 mg/kg/day for F1 and F2 generation pups.
At 1000 mg/kg/day, in F0 generation the mean number of pups after culling on lactation day 4 (9 ± 1.8, see Table 5) was lower than other dose levels due to smaller litters.


F1 Cohort 1B Animals
There were four unscheduled deaths in F1 Cohort 1B animals during the course of this study. None of these were considered to be test-item related, due to no test-item related histological findings.
At 100 mg/kg/day, Animal 2636F was euthanised on Study Day 114, due to poor condition during parturition. The gross necropsy findings noted, pale discoloration of the pituitary gland, however, the cause of this animal’s poor condition was undetermined.
At 0 mg/kg/day, 1134M was found dead on Study Day 88, there was no clinical and no gross necropsy observations associated with the animal’s death. Animal 1629F was euthanised on Study Day 113, due to clinical observations of abnormal respiratory rate, shallow breathing, abnormal breathing sounds, erected fur, brown fur straining around anus, subdued, cold to touch with a prominent backbone, pale eye colour and brown liquid material present. The gross necropsy findings noted, there were dark red foci in the thymus and dead fetuses in the uterus. The latter may have accounted for this animal’s poor condition although this cannot be stated with any certainty as tissues were not evaluated microscopically. Animal 1633F was euthanised on Study Day 76, due to clinical observations of limited usage of right forelimb, swollen forelimb, hunched posture, erected fur, abnormal gait, swollen dorsal cervical area and decreased activity. Grossly, there was a perforation in the oesophagus which was considered most likely to be attributable to a gavage accident, partly accounting for the condition and clinical signs described above.
F1 Surplus PND 52 Pups
At 1000 mg/kg/day, Animal 4209M was euthanised on Study Day 118, due to body weight loss of 10%. At 100 mg/kg/day, animal 2211M was euthanised on Study Day 129, due to left hind paw injury resulting in a swollen left hindlimb, missing digit on left hindlimb, abnormal gait, hunched posture and in self-injurious behaviour.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, in F1 and F2 generations the litter weights and pup individual weights (male and female) were slightly lower from PND 14 – PND 21, when compared with controls.
There were no test item-related effects on pup weight at dose levels of 100 and 300 mg/kg/day.

F1 Cohort 1A Animals
In Cohort 1A, males dosed at 1000 mg/kg/day had consistently lower body weights of up to 13.9% throughout dosing which were statistically significant (on Day 1, 62, 69, 75 and 76), and lower body weight gains which were significant between dosing Day 48 – 62.
The starting body weights in females, dosed at 1000 mg/kg/day, were -10.5 % lower, when compared with controls. The body weight gain of these animals was lower (Day 48 – 69), resulting in statistically significant lower mean body weight (Day 55 – 69), up to 6.7% lower, when compared with controls.
There was no effect on body weight in F1 males and F1 females, dosed at 100 and 300 mg/kg/day and were considered to be unaffected by treatment.
F1 Cohort 1B Animals
In cohort 1B, F1 males dosed at 1000 mg/kg/day, had lower body weights at the start of dosing, when compared with the controls. They had slightly lower body weight gains and statistically significant lower body weights (up to -10% relative to control) were observed from Day 55 till the end of dosing.
Similarly, F1 females dosed at 1000 mg/kg/day, had lower body weights, when compared to controls. These differences were statistically significant on Day 1 and from Day 55 during premating period (up to -8%), then throughout gestation (up to -11.5%) and lactation (up to - 14%). This correlated with lower food consumption of females during premating, gestation and lactation.
There was no test item-related effect on body weight in F1 males and F1 females, dosed at 100 and 300 mg/kg/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

F1 Cohort 1A Animals
There were no test item related effects on food consumption of F1 males and F1 females in Cohort 1A dosed at 100, 300 or 1000 mg/kg/day.
F1 Cohort 1B Animals
In Cohort 1B, during premating period females dosed at 1000 mg/kg/day had lower food consumption, mean difference up to 15.8% lower, when compared with controls.
During gestation, F1 females dosed at 1000 mg/kg/day had statistically significant lower food consumption (on Days 14-20), where the mean was up to 11.73% lower, when compared with controls.
Similarly, during lactation, F1 females dosed at 1000 mg/kg/day also had lower food consumption, which was statistically significant from Day 7 -14 (-8.6 %) and Day 14 – 21 (- 10.3 %) when compared with controls.
There were no test item-related effects on food consumption of F1 Cohort 1B females dosed at 100 or 300 mg/kg/day and F1 Cohort 1B males dosed at 100, 300 or 1000 mg/kg/day.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology:
There were no test item-related effects on hematology parameters at dose levels of 100, 300 or 1000 mg/kg/day. Differences in some haematology parameters attained statistical significance, however there was either no dose-related trend or were minor and of insufficient magnitude to be considered toxicologically relevant.
Coagulation:
In the Cohort 1A females, there was a statistically significant decrease in fibrinogen concentration at 1000 mg/kg/day. This finding was considered to be incidental and not test item-related.
There were no other test item-related effects on coagulation parameters at dose levels of 100, 300 or 1000 mg/kg/day in F0 and F1 animals.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Differences in some cases attained statistical significance, however they were minor and of insufficient magnitude to be considered toxicologically relevant.
There were no test item-related effects on clinical chemistry parameters at dose levels of 100, 300 or 1000 mg/kg/day.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

At 300 or 1000 mg/kg/day there were statistically significant lower urine pH levels in F1 Cohort 1A males (6.65 or 6.05, respectively; control 8.50) and females (6.80 or 6.30, respectively; control 8.11), when compared with controls.
There were no test item-related effects on urinalysis parameters at dose levels of 100 mg/kg/day.

Sexual maturation:

no effects observed

Description (incidence and severity):

There was no test item effect on the age of sexual maturation (vaginal opening or preputial separation).

Anogenital distance (AGD):

effects observed, non-treatment-related

Description (incidence and severity):

There was a decrease in anogenital distance in F1 male pups at 1000 mg/kg/day, this pattern was not consistent in F2 generation, where the anogenital distance increased in males dosed at 1000 mg/kg/day. These findings were not consistent across generations and were not dose-dependent therefore, were considered not to be test item-related.
There was no test item-related effect on anogenital distance in F1 and F2 pups at dose levels of 100 or 300 mg/kg/day.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

In the F1 generation, there were 5 litters (2503, 2509, 2514, 2515 and 2517) dosed at 100 mg/kg/day, 1 litter (3513) dosed at 300 mg/kg/day and 2 litters (4504 and 4516) dosed at 1000 mg/kg/day, in which male pups had retained nipples on PND 13.
In the F2 generation, there was 1 litter (2638) dosed at 100 mg/kg/day and 1 litter (3630) dosed at 300 mg/kg/day, in which male pups had retained nipples on PND 13.
There was no dose-dependent pattern observed and no correlation between the nipple retention and anogenital distance, therefore this was considered not to be test item-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Cohort 1A Animals
There were no test item-related organ weight differences.
Compared to controls, there were several statistically significant weight differences which were considered not to be test item – related. Lower group mean male adrenal gland (absolute), spleen (absolute) and kidney weights (absolute and relative to brain weight), lower female heart (absolute) and kidney weights (absolute) at 1000 mg/kg/day, along with lower group mean male heart weights (absolute and relative to brain and body weight or relative to brain weight only) at ≥ 100 mg/kg/day were all considered to be due to the lower mean terminal body weight in these groups. Although group mean female liver weights (relative to body weight) were higher at 1000 mg/kg/day (reminiscent of that seen in F0 females), when the individual animal data was examined, there were only 4/20 animals in which the body weight adjusted values were slightly higher than the control range (and only 1/20 and 2/20 animals were slightly above control ranges when absolute and brain weight adjusted values were examined, respectively). As such, this was considered insufficient evidence of a test item-related effect.
There were other organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were DPT- related.
F1 Cohort 1B Animals
There were no test item-related organ weight differences.
Compared to controls, there were statistically significant weight differences which were considered not to be test item-related. Higher group mean seminal vesicle weight (relative to body weight) in males at 1000 mg/kg/day was considered to be due to the lower mean terminal body weight in this group. Lower group mean thymus weight (absolute) was noted in females at 1000 mg/kg/day. However, given that when the individual animal data was examined there were only 3/25 animals in which the absolute value was slightly lower than the control range (and only 2/25 animals were slightly below the control range for body weight adjusted values) there was considered insufficient evidence of a test item-related effect.
There were other organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were DPT-related.
F1 Unselected PND 22-24 Pups
There were no test item-related organ weight differences.
Compared to controls, there were several statistically significant weight differences which were considered not to be test item – related. Higher group mean male and female brain weights (relative to body weight) at 1000 mg/kg/day, lower group mean male liver weight (absolute) at 1000 mg/kg/day and lower group mean female liver weights (absolute and relative to brain weight) at ≥ 300 mg/kg/day were considered to be due to the lower mean terminal body weights in these groups.
There were other organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were DPT-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Cohort 1A Animals
There were no test item-related gross findings.
A 15x12x5 mm soft, dark red mass evident grossly in the mandibular salivary gland of 2603F dosed at 100 mg/kg/day was also considered noteworthy. However, given that only a single animal dosed at 100 mg/kg/day was affected it was considered not to be test item – related.
All remaining gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be DPT-related.
F1 Cohort 1B Animals
Animal 4143M was found not to have testis or epididymis at necropsy, however this animal was paired with 4645F for 2 nights before a positive mating sign of an oestrous vaginal smear with sperm was recorded, the female then produced a litter of 12 pups as a result of this mating. The organ weights of the semi vesicle, prostate gland, pituitary gland, and thymus were within the range of the controls, thus showing no indication of any possible malformation. Therefore, it is likely that 4143M had testis and epididymis at necropsy but were missed in error.
There were no test item-related gross findings.
The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be DPT-related.
F1 Unselected PND 22-24 Pups
There were no test item-related gross findings. Dark red discolouration was noted in the thymus of a single male pup (3309M) dosed at 300 mg/kg/day. This is a common incidental finding typically associated with agonal haemorrhage and was considered not to be DPT-related.
F1 PND 52 Surplus Pups
There were no test item-related gross findings
The gross findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be DPT-related.

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F1 Cohort 1A Animals
There were no test item-related microscopic findings.
An area of moderate agonal haemorrhage was evident in the mandibular salivary gland of 2603F correlating with the dark red mass noted grossly; it was considered not to be test item – related.
The remaining microscopic findings observed were considered incidental, of the nature commonly observed in this strain and age of rat or occurred at a similar incidence in control and treated animals, and, therefore, were considered not to be DPT-related.

Other effects:

no effects observed

Description (incidence and severity):

Endocrine findings:
There were no test item related effects on TSH and T4 at dose levels of 100, 300 or 1000 mg/kg/day.


Immunophenotyping:
There were no test item related effects were observed as illustrated by comparison of immunophenotypic expression in rat splenocytes between animals dosed with 100, 300 or 1000 mg/kg/day and the control group, dosed with 0 mg/kg/day. No dose dependent or sex related effects were observed with regards to any immune cell populations, as percentage of total lymphocytes were comparable between animals dosed with 100, 300 or 1000 mg/kg/day and the control group.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no test item-related effects on litter survival, viability index or lactation index at dose levels of 100, 300 or 1000 mg/kg/day for F1 and F2 generation pups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, in F1 and F2 generations the litter weights and pup individual weights (male and female) were slightly lower from PND 14 – PND 21, when compared with controls.
There were no test item-related effects on pup weight at dose levels of 100 and 300 mg/kg/day.

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

There was a decrease in anogenital distance in F1 male pups at 1000 mg/kg/day, this pattern was not consistent in F2 generation, where the anogenital distance increased in males dosed at 1000 mg/kg/day. These findings were not consistent across generations and were not dose-dependent therefore, were considered not to be test item-related.
There was no test item-related effect on anogenital distance in F1 and F2 pups at dose levels of 100 or 300 mg/kg/day.

Nipple retention in male pups:

effects observed, non-treatment-related

Description (incidence and severity):

In the F2 generation, there was 1 litter (2638) dosed at 100 mg/kg/day and 1 litter (3630) dosed at 300 mg/kg/day, in which male pups had retained nipples on PND 13.
There was no dose-dependent pattern observed and no correlation between the nipple retention and anogenital distance, therefore this was considered not to be test item-related.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

F2 Unselected PND 22-24 Pups
There were no test item-related organ weight differences.
Compared to controls, the statistically significant higher group mean male liver weight (relative to body weight) at 300 mg/kg/day was considered not to be test item-related, given the lack of dose relationship.
There were other organ weight values that were different from their respective controls. There were, however, no patterns or correlating data to suggest these values were DPT- related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

F2 Unselected PND 22-24 Pups
There were no test item-related gross findings. Dark red foci were noted in the thymus of two female pups (4908F and 4909F) dosed at 1000 mg/kg/day. This is a common incidental finding typically associated with agonal haemorrhage and was considered not to be DPT-related.

Effect levels (F2)

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F2)

Critical effects observed:

no

Overall reproductive toxicity

Reproductive effects observed:

no

Any other information on results incl. tables

Summary Group Mean Organ Weight Data – Scheduled Euthanasia F0 Females

	Females
Group	1	2	3	4
Dose (mg/kg/day)	0	100	300	1000
No. animals per group	25	24	25	22
Liver (No. weighed)	(25)	(24)	(25)	(22)
Absolute value (g)	17.2	18.0	17.9	19.5^**
% of brain weight	839	884	865	947^*
% of body weight	5.11	5.33	5.23	5.83^**
Thymus (No. weighed)	(25)	(24)	(25)	(22)
Absolute value (g)	0.205	0.207	0.190	0.162^*
% of brain weight	10.0	10.2	9.2	7.8^*
% of body weight	0.06	0.06	0.06	0.05

Anova & Dunnett: ^* = p≤0.05; ^** = p≤0.01

 

Summary Reproductive Performance – F0 Generation  

Group/ Dose Level (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (400)
Male Mating Index	1.00	1.00	1.00	1.00
Male Fertility Index	0.92	0.96	1.00	1.00
Male Pregnancy Index	0.92	0.96	1.00	1.00
Female Mating Index	1.00	1.00	1.00	1.00
Female Fertility Index	0.92	0.96	1.00	1.00
Female Pregnancy Index	0.92	0.96	1.00	1.00
Mean number of nights before positive mating sign	2.6	2.9	2.2	2.8

Text Table 38
Summary Reproductive Performance – F1 Generation  

Group/ Dose Level (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (400)
Male Mating Index	1.00	1.00	1.00	1.00
Male Fertility Index	0.96	0.84	1.00	0.96
Male Pregnancy Index	0.96	0.84	1.00	0.96
Female Mating Index	1.00	1.00	1.00	1.00
Female Fertility Index	0.96	1.00	1.00	1.00
Female Pregnancy Index	0.96	0.84	1.00	0.96
Mean number of nights before positive mating sign	2.3	2.8	2.5	2.9

 

 

Summary Litter Data – F0 Generation  

Group/ Dose Level (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (400)
Mean number of implant sites per pregnancy ± standard deviation	17±2.2	16 ±2.5	16 ±1.4	15 ±2.6
Mean total number of pups born per litter ± standard deviation	16 ± 2.7	14 ± 4.2	14 ± 2.2	13 ± 5.2
Viability Index Days 0-4	97	99	99	97
Lactation Index Days 4-21	100	100	100	96

Text Table 40
Summary Litter Data – F1 Generation  

Group/ Dose Level (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (400)
Mean number of implant sites per pregnancy ± standard deviation	17 ± 2.5	16 ± 1.8	16 ± 2.0	16 ± 1.7
Mean total number of pups born per litter ± standard deviation	16 ± 2.7	13 ± 4.5	15 ± 2.3	14 ± 2.3
Viability Index Days 0-4	94	97	98	100
Lactation Index Days 4-21	100	100	100	100

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of Di (iso) pentyl terephthalate (DPT), by once daily oral gavage was well tolerated in male and female rats at dose levels of up to the limit dose of 1000 mg/kg/day, with no effects on development and reproductive function.
Based on the results of this extended one generation reproductive toxicity study (Cohort 1A, Cohort 1B), the following no-observed-adverse-effect level (NOAEL) of DPT were established:
Parental toxicity (F0 and F1): 1000 mg/kg/day
Reproductive NOAEL (F0 and F1): 1000 mg/kg/day
Post-Natal Developmental NOAEL (F1 and F2): 1000 mg/kg/day

Executive summary:

The objective of this study was to determine the potential toxicity of the chemical Di‑(iso)‑pentyl terephthalate (DPT), an industrial chemical, when given by oral (gavage) to adult rats and their offspring. This study was designed to provide an evaluation of the pre and post natal effects on development as well as systemic toxicity in pregnant and lactating females and young and adult offspring. Detailed examination of key developmental endpoints, such as offspring viability, neonatal health, developmental status at birth, physical and functional development until adulthood was expected to identify specific target organs in the offspring. In addition, the study aimed to provide information about the effects of DPT on the integrity and performance of the adult male and female reproductive systems.

The following parameters and end points were evaluated in this study: mortality, clinical observations, body weights, food consumption, estrous cycles and reproductive performance, observations of females with litters, pre‑weaning physical development of F1 and F2 pups, milk analysis, clinical pathology parameters (haematology, coagulation, clinical chemistry, urinalysis, Thyroid Stimulating Hormone [TSH] and Thyroxine [T4] analysis, gross necropsy findings, organ weights, ovarian follicle counts, sperm evaluation (F0 and Cohort 1A males), immunophenotyping and histopathological examinations.

There were no unscheduled deaths in this study that were considered to be test item-related.

Administration of DPT was associated with clinical observations of higher incidence of ploughing and salivation immediately to 1-hour post dose and were noted in both F0 and F1 animals at all dose levels, but particularly at 300 and 1000 mg/kg/day. These findings were considered to be most likely a result of the taste and formulation of the test item, therefore, were considered not to be adverse. In the F1 Cohort 1B males and females were noted to have ungroomed fur, at all dose levels, however these findings were only noted in this cohort, therefore were not reproducible and considered not adverse.

Additionally, administration of DPT at 1000 mg/kg/day was associated with slightly lower body weight in both males and females. In F0 males at 1000 mg/kg/day, had slightly lower body weight gains with sporadic statistical significance, resulting in lower mean bodyweights on Day 71 (-3.7%) and on Day 113 (-5.2%), when compared with controls.  In F0 females at 1000 mg/kg/day, there were lower bodyweights during premating period (-4.7%), when compared with controls and during gestation had lower mean bodyweight gains GD 14 - 20 ( -20%), when compared with controls. This changed during lactation period where the females at 1000 mg/kg/day, had higher body weight gains however, the mean body weights remained slightly lower on LD 21 (-2%), when compared with controls. The lower body weights were also observed in F1 males and females dosed at 1000 mg/kg/day. The F1 Cohort 1A males had lower weights up to -13.5% and females had lower body weights at the start of dosing (-10.5 %) this remained throughout the study resulting in up to 6.7% lower mean body weights (Day 55 – 69). The F1 Cohort 1B males had lower body weight gains observed from Day 55 until the end of dosing resulting in lower body weights (up to -10% relative to control). Furthermore, the F1 Cohort 1B females had lower body weights than the controls and this maintained throughout gestation and lactation where the mean body weights were up to -14% reduced, when compared with controls. The lower body weights in the females correlated with the lower food consumption in F0 and Cohort 1B females. The overall mean body weight did not exceed 10% below of starting weights or controls throughout dosing, therefore was considered not adverse.

There were no test-item effects on F0 and F1 generation reproduction performance: fertility, mating, estrous cycle or sperm parameters.

At 1000 mg/kg/day, in F1 and F2 the pup weights (male and female) were slightly lower from PND 14 – PND 21, when compared with controls. This was considered to be a result of lower bodyweight and food consumption of females during gestation and lactation. The nipple retention noted in some litters in F1 and F2 generation was considered not to be test item-related because there was no dose-depended pattern observed and no correlation to anogenital distance. There were no other test-item related effects on pup development parameters: litter survival, live birth index, viability index, lactation index, anogenital distance or sexual maturation of the pups.

There were no test-item related effects on thyroid analysis, haematology or clinical chemistry parameters. In the Cohort 1A females, there was a statistically significant decrease in fibrinogen concentration at 1000 mg/kg/day. This finding was considered to be incidental and not test item-related.

There were lower urine pH levels, in F0 and F1 Cohort 1A males and females dosed at 300 or 1000 mg/kg/day and F0 females dosed at 1000 mg/kg/day had lower specific gravity values, however these were considered not adverse in the absence of kidney related findings.

There were no test item-related, dose- or sex-depended effects on any splenocyte immune cell populations.

There were no test item-related gross findings or microscopic findings. However, test item-related higher organ weight were noted in the liver and thymus of F0 female dosed at 1000 mg/kg/day.

In conclusion, administration of Di‑(iso)‑pentyl terephthalate (DPT), by once daily oral gavage was well tolerated in male and female rats at dose levels of up to the limit dose of 1000 mg/kg/day, with no effects on development and reproductive function.

Based on the results of this extended one generation reproductive toxicity study (Cohort 1A, Cohort 1B), the following no-observed-adverse-effect level (NOAEL) of DPT were established:

Parental toxicity (F0 and F1): 1000 mg/kg/day 

Reproductive NOAEL (F0 and F1): 1000 mg/kg/day                                

Post-Natal Developmental NOAEL (F1 and F2): 1000 mg/kg/day