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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
one-generation reproductive toxicity
Remarks:
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 27 to May 18, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
27 July 1995
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): diallyl phthalate
- Physical state: clear colourless liquid
- Storage condition of test material: 4°C in the dark, under nitrogen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: Sprague-Dawley Crl:CD (SD) IGS BR
- Source: Charles River (UK) Limited, Manston Road, Margate, Kent
- Weight at study initiation: (P) Males: 308-366 g; Females: 196-234 g
- Housing: 5 rats/sex in polypropylene cages with stainless steel grid floors and tops, suspended over paper-lined polypropylene trays
- Diet: pelleted diet ad libitum
- Water: ad libitum
- Acclimation period: 16 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 55
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on mating procedure:
- M/F ratio per cage: 1/1
- Proof of pregnancy: vaginal plug / sperm in vaginal smear
- After successful mating each pregnant female was caged (how): individually in polypropylene cage with solid floors and stainless steel tops, and given softwood chips as bedding
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Verification by HPLC
Nominal concentrations used: 3.3, 10 & 30 mg/ml (test substance/vehicle)
Mean concentration found (% of nominal): 3.26 (99), 10.3 (103) & 39.7 (99) mg/ml
Duration of treatment / exposure:
Throughout maturation, mating, gestation and up to day 4 post partum.
Frequency of treatment:
Once daily
Details on study schedule:
Following 14 d of exposure, rats were allowed to mate and offspring was followed for up to 5 d post partum when all animals were killed.
Doses / concentrations
Remarks:
Doses / Concentrations:
16.7, 50 & 150 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
10 rats/sex/dose
Control animals:
yes, concurrent vehicle
Positive control:
Not included.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: each morning
- Cage side observations: ejected copulation plugs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily, immediately before dosing, immediately after dosing and 1 hr after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the maturation phase for all animals and continued for males after the mating phase, and for mating females: on days 0, 7, 14 & 20 post-coitum and days 1 & 4 post-partum

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Consumption recorded between days 1 to 7, 7 to 14 and 14 to 20 post coitum and day 1 to 4 post partum.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: [yes/no]
- If yes, maximum of [...] pups/litter ([...]/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, other: clinical conditions, individual litter weights, detachment of pinna, surface righting reflex

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals, 4 days post-partum
- Maternal animals: All surviving animals, 4 days post-partum

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated were prepared for microscopic examination and weighed, respectively: coagulating glands, epididymes, prostate, seminal vesicles, testis, liver, pituitary, ovaries, uterus/Cervix, vagina, stomach
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed at 5 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: external and internal abnormalities
Statistics:
- Adult bodyweight, food consumption, litter size, litter weight, individual offspring bodyweight, offspring landmarks of physical development, reproductive and viability indices and adult organ weights: Bartlets chi-square test followed by one-way analysis of variance, followed by a t-test (if unequal variances) or the Dunett's Multiple Comparison Method (if equal variances)
- Adult pre-coital intervals, gestation lengths, offsprinf reflexological responses, litter sex ratios, relative organ weights: Kruskal-Wallis non-parametric rank sum test, followed by a Mann-Whitney 'U' test (if significant differences)
Histopathology: Chi-square analysis, Kruskal-Wallis one-way non-parametric analysis of variance
Reproductive indices:
Mating index (%) = (Nb of animals mated/Nb of animals paired) x 100
Pregnancy index (%) = (Nb of pregnant females/Nb of animals mated) x 100
Parturition index (%) = (Nb of females delivering live pups/Nb of pregnant females) x 100
Offspring viability indices:
Live birth index (%) = (Nb of pups alive/Nb of pups born) x 100
Viability index 1 (%) = (Nb of pups alive on Day 4/Nb of pups alive on Day 1) x 100
Viability index 2 (%) = (Nb of pups alive on Day 5/Nb of pups alive on Day 4) x 100
Viability index 3 (%) = (Nb of pups alive on Day 5/Nb of pups alive on Day 1) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
associated with suspected dystocia

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS):
- All doses: increased salivation immediately post dose in all animals (150 and 50 mg/kg) or in isolated incidents (16.7 mg/kg).
- 150 mg/kg: 2 females killed in extremis and one female died, probably due to dystocia (an increase in gestation length). All pups or foetusses were alive except for one canabilised.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- Reduction of mean body weight at 150 mg/kg bw, but not statistically significant compared to the control group. No effects on body weights at the lower doses.
- No treatment related increase of food consumption at 60 and 150 mg/kg bw.

MATING PERFORMANCE:
- No treatment related effects.

GESTATION LENGTH AND PARTURITION:
- 150 mg/kg bw: 3 out of 10 females showed a delayed parturition, which may be attributed to the dosage applied. No delay was recorded for the other females at this dose or at the lower doses.

MACROSCOPIC FINDINGS:
- 150 mg/kg: in 50% males and 90% females areas of patchy pallor and/or accentuated lobular pattern on the liver and in one female also ulceration of the glandular region of the stomach with digested blood.
- No significant differences in the number of corpora lutea, implantation sites or offspring viabililty between groups
- organ weights: only absolute epidydimides weights at 150 mg/kg bw were significantly lower compared to the controls.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- 150 mg/kg bw: statistically sigificant occurrence of periportal hepatocyte necrosis, enlargement and basophilia, bile duct proliferation and periportal fibrosis. No treatment related findings at lowes doses.

Effect levels (P0)

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: clinical signs; mortality; histopathology

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: other: viability, growth and development from conception to early lactation

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study in rats, administration of diallyl phthalate to rats throughout maturation, mating, gestation and early lactation phases of reproduction did not induce any treatment related effects on fertility or mating performance. Mortality and extreme effects recorded in three females prior to parturition indicated a possible effect on gestation length. There was no effect on offspring viability, growth and development. The NOAEL for general and reproductive toxicity in adults is 50 mg DAP/kg bw and the NOAEL for fertility and offspring is 150 mg DAP/kg bw.
Executive summary:

In an oral study in rats by OECD reproduction/developmental toxicity screening test [OECD TG 421], rats (10animals/sex/group) were dosed by gavage at 0, 16.7, 50 and 150 mg/kg bw/day from 14 days prior to mating today 4 of lactation. At 150 mg/kg/day there were 3 mortalities, which were associated with possible dystocia.

Effects on newborns and live newborns were not evaluated in these female rats. Histopathological changes in theliver of parental animals were observed at this dose. There were no treatment-related effects on the fertility ofmale or female rats. No treatment-related histopathological changes were found in the reproductive organs ofparental animals. There were no treatment-related effects on offspring viability, growth and development from conception to early lactation. No morphological abnormalities were seen in offspring of rats given this chemical

pre- and postnatally. The NOAELs for general toxicity in parent animals and for female reproductive toxicity were 50 mg DAP/kg bw/day. The NOAEL for fertility and offspring was 150 mg DAP/kg bw.