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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18-02-2013 to 09-09-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Justification for study design:
Screening test for reproduction and development.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Colour: colourless
- Odour: odourless
- CAS-Number: 89-32-7
- Molecular formula: C10 H2 O6
- Molecular weight: 218.12
Specific details on test material used for the study:
PMDA, pyromellitic dianhydride 99.94%; batch NJC1207029

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Han™:RccHan™:WIST
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
The animals were acclimatised for seven days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 298 to 350g, the females weighed 198 to 222g, and were approximately twelve weeks old.
Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding. During the pairing phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes.
The animals were allowed free access to food and water. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Appendix 21. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidity’s are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2 °C and 55 ± 15 % respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
Carboxymethyl cellulose (1%) & Tween 80 (0.1%).
Details on exposure:
Oral gavage daily through the study until termination.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC methodology. Previous studies have shown that the test substance is stable in the vehicle and homogeneous.
Concentrations across all dosages were in the range 88% to 111% with the majority of values at or above 100%.
Duration of treatment / exposure:
Throughout pre-mating, pairing, gestation and the post-partum phase.
Frequency of treatment:
Oral gavage daily through the study until termination.
Details on study schedule:
The study was performed to screen for potential adverse effects of the test item on reproduction, including offspring development, and provides a hazard assessment for potential effects on reproduction.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
12 males and 12 females.
Control animals:
yes, concurrent vehicle
Details on study design:
Chronological Sequence of Study
i. Groups of twelve male and twelve female animals were treated daily at the appropriate dose level throughout the study (except for females during parturition where applicable). The first day of dosing was designated as Day 1 of the study.
ii. On Day 15, animals were paired on a 1 male: 1 female basis within each dose group for a maximum of fourteen days.
iii. Following evidence of mating (designated as Day 0 coitum) the males were returned to their original cages and females were transferred to individual cages.
iv. Pregnant females were allowed to give birth and maintain their offspring until Day 5 post partum. Litter size, offspring weight and sex, surface righting and clinical signs were also recorded during this period.
v. The male dose groups were killed and examined macroscopically on Day 43.
vi. At Day 5 post partum, all females and surviving offspring were killed and examined macroscopically.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, soon after dosing, and one hour and five hours after dosing during the working week. Animals were observed immediately before dosing, soon after dosing and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.

Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 p coitum, and Animals were also weighed at termination.

During the pre-pairing period, weekly food consumption was recorded for each cage of adults until pairing. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded during the lactation period (Days 1-4).
Weekly food efficiency (body weight gain/food intake) was calculated retrospectively for males and for females during the pre-pairing phase. Due to offspring growth and milk production for lactation, food efficiency for females could not be accurately calculated for females during gestation and lactation.

Water intake was observed daily by visual inspection of water bottles for any overt changes.
Oestrous cyclicity (parental animals):
Not formally assessed. But the Pre-Coital Interval data suggest no disturbance in estrous cyclicity.
Sperm parameters (parental animals):
Assessed during histopathological examination.
Litter observations:
Litter Data
On completion of parturition (Day 0 post partum),the number of live and dead recorded. Offspring were individually identified within each litter by tattoo on Day 1 post partum.
For each litter the following was recorded:
i. Number of offspring born
ii. Number of offspring alive recorded daily and reported on Days 1 and 4 post partum
iii. Sex of offspring on Days 1 and 4 post partum
iv. Clinical condition of offspring from birth to Day 5 post partum
v. Individual offspring weights on Days 1 and 4 post partum (litter weights were calculated retrospectively from this data)

Physical Development
All live offspring were assessed for surface righting reflex on Day 1 post partum.
Postmortem examinations (parental animals):
Adult males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 5 partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy or produce a litter were killed on or after Day 25 post coitum.
For all females, the uterus was examined for signs of implantation and the number of uterine implantations in each hom was recorded. This procedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). The corpora lutea were also counted.
All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs were removed from terminal kill adult animals, dissected free from fat and weighed:
Adrenals
Brain
Epididymides
Heart
Kidneys
Liver
Ovaries
Pituitary (post-fixation)
Prostate
Seminal vesicles Spleen Testes Thymus
Thyroid/parathyroid (post fixation)

Histopathology
Samples of the following tissues were preserved from all animals from each dose group:
Coagulating gland, Epididymides, Ovaries ,Mammary gland, Prostate, Seminal vesicles, Testes, Uterus/Cervix, Pituitary,Vagina, Gross Lesions
Additional tissues were preserved from five males and five females from each dose group in buffered 10% formalin.
Postmortem examinations (offspring):
Interim deaths were exanined at necropsy.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Body Weight, Body Weight Change, Food Consumption during gestation and lactation, PreCoital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righting, Absolute Organ Weights, Body Weight- Relative Organ Weights.
Reproductive indices:
Pre-coital Interval, Fertility Indices: mating index, pregnancy index; Gestation length, Patrurition index.
Offspring viability indices:
Implantation Losses (%); Live Birth and Viability Indices: Live Birth Index (%); Number of offspring born; Number of offspring alive on Day 4; Viability Index (%); Number of offspring alive on Day 1; Sex Ratio (% males).

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No clinically observable signs of toxicity were detected during the study. Clinical signs were confined to the presence of post-dose increased salivation for ten male and ten female animals of either sex treated with 750 mg/kg bw/day.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A slight reduction in body weight gains was evident for males treated with 750 mg/kg bw/day during the first two weeks of treatment, with statistically significant differences detected during the first week in comparison to control values (p<0.05). This resulted in a lower overall body weight gain for the study period at 750 mg/kg bw/day in comparison to the concurrent control group.
Reduced body weight gains were also noted for females treated with 750 mg/kg bw/day during the first two weeks of treatment, although statistical significance was only achieved during Week 1 when compared to controls. There was no difference in body weight change detected during the gestation phase for treated females in comparison to controls.
There were no further differences in body weight change considered to be a result of treatment with the test item.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Males treated with 750 mg/kg bw/day showed an increase in thyroid/parathyroid weights when compared to controls, with a number of absolute and relative values outside of the normally expected ranges.
Females treated with 750 mg/kg bw/day showed an increase in absolute and relative liver weights when compared to controls (p<0.05) with one higher than expected absolute and relative value.
There were no further organ weight changes which were considered to be related to treatment with the test item.
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Pre-Coital Interval data suggest no disturbance in estrous cyclicity
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No evidence from the histopathological examination of the reproductive organs.
Reproductive performance:
no effects observed

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: No adverse toxicity.

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
A very small number of interim deaths showing no relationship to treament.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No effects on litter weights, offspring weights or weight gain.
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
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
Other effects:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No treatment toxicity was evident.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
750 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse toxicity.
Remarks on result:
other: No adverse toxicity.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

Conclusions:
Treatment of rats in an OECD 421 study with PMDA ( pyromellitic dianhydride ) had no adverse effects apart from an effect on body weight at 750 mg/kg bw/day. Importantly, there were no adverse efftects on reproductive performance, mating, fertility, gestation, parturition, or on F1 offspring survival or development.
Executive summary:

Twelve male and twelve female rats, were treated for up to eight weeks (including a two week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 50, 250 and 750 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle alone. Clinical signs, body weight change, food and water consumption were monitored during the study.

Pairing of animals within each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear their offspring to Day 5 of lactation. During the lactation phase., daily clinical observations were performed on all surviving offspring, together with litter size and offspring weights and assessment of surface righting reflex.

Adult males were sacrificed or, Day 43, followed by the sacrifice of all females and offspring on Day 5 of lactation. Any female which did not produce a pregnancy was terminated on or after Day 25post coitum.All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

Apart from an effect on body weight at 750 mg/kg bw/day no other treatment related effects were evident.

PMDA ( pyromellitic dianhydride ) had no adverse efftects on reproductive performance, mating, fertility, gestation, parturition, or on F1 offspring survival or development.