Registration Dossier

Administrative data

Endpoint:
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
Remarks:
Reliability 1

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 421 screening for reproduction and development
Deviations:
no
GLP compliance:
yes
Limit test:
no

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 Wistar Han™:RccHan™:WIST
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 appro
ximately 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 origi
nal cages. Mated females were housed individually during gestation and lactation in solid floor pol
ypropylene 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 polyc
arbonate bottles attached to the cage.

The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., S
hardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air c
hanges per hour and the low intensity fluorescent lighting was controlled to give twelve hours cont
inuous 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 randomi
sation 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:
CMC (carboxymethyl cellulose) Carboxymethyl cellulose (1%) & Tween 80 (0.1%).
Details on exposure:
Oral gavage daily through the study until termination.
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%.
Details on mating procedure:
Animals were paired on a 1 male: 1 female basis within each dose group, for a period of up to fourte
en 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 ev
idence of mating (Day 0 of gestation) and the males were subsequently returned to their original ho
lding cages. Mated females were housed individually during the period of gestation and lactation.
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.
Duration of test:
Throughout pre-mating, pairing, gestation and the post-partum phase.
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 vwelve 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 maxim
um 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.

Examinations

Maternal 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 w
eekends 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 fe
males 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.

Estrous 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 retrospect
ively 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 exsang
uination on Day 43. Adult females were killed by intravenous overdose of sodium pentobarbitone foll
owed by exsanguination on Day 5 partum. Surviving offspring were terminated via intracardiac overdo
se 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 im
plantations 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 we
ighed:
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.
Ovaries and uterine content:
Examined at terminal necropsy.
Fetal examinations:
Neonates examined at terminal 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 I
nterval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea, Implantation Sites,
Implantation Losses, Viability Indices, Offspring Body Weight, Offspring Body Weight Change, Offs
pring Surface Righting, Absolute Organ Weights, Body Weight- Relative Organ Weights.
Indices:
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).
Historical control data:
Available for appropriate comparisons.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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 t
he 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):
Females treated with 750 mg/kg bw/day showed an increase in absolute and relative liver weights wh
en 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

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
LOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
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.
Basis for effect level:
body weight and weight gain

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
Neonatal weights.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Neonates.
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
not examined
Visceral malformations:
not examined
Other effects:
no effects observed

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No developmental effects at high dose

Overall developmental toxicity

Key result
Developmental 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. All animals were subjected to a gross necropsy examination and histopathological evaluation of reproductive tissues was performed.

 

The only sign of toxicity was an effect on body weight at 750 mg/kg bw/day. Moreover, pre- and post-implantation was unaffected as was parturition. Live birth and viability indices were also unaffected as was sex ratio, survival and development of the offspring. PMDA had no effect on the development of the foetuses or live offspring.

 

It can be concluded that pyromellitic dianhydride (PMDA) had no adverse effects on reproductive performance, mating, fertility, gestation, development of the foetus, parturition, or on F1 offspring survival or development.