Reaction mass of bis(oxiran-2-ylmethyl) terephthalate and tris(oxiran-2-ylmethyl) benzene-1,2,4-tricarboxylate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

Preliminary Study of Reproductive Performance

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 June 2018 - 19 October 2018

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

other: Preliminary Study of Reproductive Performance

Justification for type of information:

The initial preliminary study was initiated in June 2018, however due to complications was terminated and a second preliminary study had to be conducted to allow sufficient information neccessary for the main Extended One Generation Study requested by ECHA.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Principles of method if other than guideline:

- Principle of test: The purpose of this study was to assess the influence of Araldite PT 910 on reproductive performance when administered orally (gavage) to CD rats, and to establish suitable dose levels for an extended one-generation reproductive performance study.

- Short description of test conditions:
Route: Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at: Constant doses in mg/kg/day.
Volume dose: 2.5 mL/kg body weight.
Individual dose volume: Calculated from the most recently recorded scheduled body weight.
Control (Group 1): Vehicle at the same volume dose as treated groups.
Frequency: Once daily at approximately the same time each day. Animals were not dosed if parturition was in progress at the scheduled time of administration.
Formulation: A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found. Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

GLP compliance:

no

Justification for study design:

Animal Model:
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Crl:CD(SD) was used because of the historical control data available at this laboratory.

Route of Administration:
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection:
A previous study of reproductive performance in the CD rat was terminated after seven weeks due to treatment related effects. Dose levels of 50, 100 and 200 mg/kg/day were used and with the exception of one female receiving 200 mg/kg/day that failed to mate, all treated F0 females failed to litter and were confirmed not to be pregnant at necropsy with no sign of any implantations. All Control females, with the exception of one female that was found dead on Day 15 of treatment, gave birth to a live litter. Signs of abnormal flattened gait were observed in all males and some females receiving 200 mg/kg/day. Six out of eight males receiving 200 mg/kg/day also showed signs of reduced muscle tone in the hind limbs and two of these males showed limited use of their hind limbs. Sperm analysis data revealed that when compared with Controls, treated males showed a dosage-related lowering of sperm motility, normal morphology and cauda epididymal sperm numbers. These effects were severe at 100 or 200 mg/kg/day. A similar pattern of effects was apparent in many sperm motion parameters with concomitant increases in % static sperm. The abnormality most commonly observed in all treated groups was decapitate sperm. Results suggested that the adverse effects occurred within the epididymides causing sperm degeneration.
The objective of the previous study was not met, therefore, in order to identify a suitable high dose level for the extended one-generation reproductive performance study, the high dose level was set at 30 mg/kg/day with intermediate and low dose levels of 15 and 3 mg/kg/day.

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate; 1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

EC Number:

940-592-6

Molecular formula:

-

IUPAC Name:

1,2,4-tris[(oxiran-2-yl)methyl] benzene-1,2,4-tricarboxylate; 1,4-bis[(oxiran-2-yl)methyl] benzene-1,4-dicarboxylate

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: AAF1344600
- Expiration date of the batch: 15 November 2018
- Purity: 82.0 - 87.0 %
- Appearance: White, solid (pellets).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature (15 to 25C) in the dark.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Males 75 to 79 days old, Females 68 to 75 days old
- Weight at study initiation: Males 377 to 430 g, Females 231 to 274 g.
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: Five days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark

IN-LIFE DATES: From: 13 June 2018 To: 21 September 2018

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was ground in a mortar using a pestle to a fine powder and mixed with a small amount of the vehicle to form a paste. Any agglomerates were broken down. Further amounts of vehicle were gradually added and mixed to produce a smooth, pourable suspension. The suspension was transferred to a measuring cylinder which had been wetted with vehicle, the mortar was rinsed with vehicle and this was added to the measuring cylinder. Vehicle was added to achieve the final volume and the suspension was transferred to a beaker and mixed using a high shear homogenizer. The suspension was transferred to the final containers, via syringe whilst magnetically stirring.
A series of formulations at the required concentrations were prepared by dilution of individual weighing of the test item.

VEHICLE
- Concentration in vehicle: 0, 1.2, 6, 12 mg/mL
- Amount of vehicle (if gavage):
- Lot/batch no. (if required):
- Purity:

Details on mating procedure:

- Pairing commenced: After a minimum of two weeks of treatment
- M/F ratio per cage: 1:1
- Length of cohabitation: Up to two weeks
- Proof of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear
- Male/female separation: Day when mating evidence was detected

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

F0 animals: For two weeks before pairing until termination after litters are weaned.
F1 animals: From weaning until Week 8 of age (after attainment of sexual maturity).
Direct treatment of F1 offspring commences at weaning (Day 21 of age).

Frequency of treatment:

Once daily at approximately the same time each day

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

F0 and F1 generation

Doses / concentrations 2

Dose / conc.:

3 mg/kg bw/day (actual dose received)

Remarks:

F0 and F1 generation

Doses / concentrations 3

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Remarks:

F0 and F1 generation

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Remarks:

F0 and F1 generation

No. of animals per sex per dose:

F0 generation: 8 animals per sex for all dose groups.
F1 generation:
for dose group 1, 2 & 3: 10 animals per sex
for dose group 4: 5 animals per sex

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for Dose Level Selection
A previous study of reproductive performance in the CD rat was terminated after seven weeks due to treatment related effects. Dose levels of 50, 100 and 200 mg/kg/day were used and with the exception of one female receiving 200 mg/kg/day that failed to mate, all treated F0 females failed to litter and were confirmed not to be pregnant at necropsy with no sign of any implantations. All Control females, with the exception of one female that was found dead on Day 15 of treatment, gave birth to a live litter. Signs of abnormal flattened gait were observed in all males and some females receiving 200 mg/kg/day. Six out of eight males receiving 200 mg/kg/day also showed signs of reduced muscle tone in the hind limbs and two of these males showed limited use of their hind limbs. Sperm analysis data revealed that when compared with Controls, treated males showed a dosage-related lowering of sperm motility, normal morphology and cauda epididymal sperm numbers. These effects were severe at 100 or 200 mg/kg/day. A similar pattern of effects was apparent in many sperm motion parameters with concomitant increases in % static sperm. The abnormality most commonly observed in all treated groups was decapitate sperm. Results suggested that the adverse effects occurred within the epididymides causing sperm degeneration.
The objective of the previous study was not met, therefore, in order to identify a suitable high dose level for the extended one-generation reproductive performance study, the high dose level was set at 30 mg/kg/day with intermediate and low dose levels of 15 and 3 mg/kg/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least twice daily

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Day 1) and weekly thereafter.
On the day of necropsy.

F0 females: Before dosing on the day that treatment commenced (Day 1) and weekly before pairing.
Days 0, 7, 14 and 20 after mating.
Days 1, 4, 7, 14 and 21 of lactation.
On the day of necropsy.

OTHER:

Signs Associated with Dosing:
Detailed observations were performed on F0 generation animals to establish and confirm a pattern of signs in association with dosing according to the following schedule:
F0 males: Week 1 - daily
Week 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week

F0 females: Week 1 - daily
Week 2 - twice (middle and end of the week)
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7, 14 and 20

Detailed observations were recorded at the following times in relation to dose administration:
Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day

Oestrous cyclicity (parental animals):

Estrous Cycle
Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs.
Wet smears: After pairing until mating, using pipette lavage.

Sperm parameters (parental animals):

Parameters examined in F0 male parental generations: sperm motility, sperm morphology, sperm count, Homogenization-resistant spermatids count

Litter observations:

Signs Associated with Dosing:
Detailed observations were performed on F1 generation animals to establish and confirm a pattern of signs in association with dosing according to the following schedule:
Selected F1 generation: Week 1 - daily
Week 2 to 4 - twice weekly (middle and end of the week)
Week 5 onwards - once each week

F1 generation: Days 21 of age to formal F1 start - daily

Body Weight:
Selected F1 generation: Twice weekly from nominal four weeks of age to termination at approximately eight weeks of age

Records Made During Littering Phase
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to maternal treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.

Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1-21 of age.
On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving, whenever possible, five male and five female offspring in each litter.

Sex ratio of each litter: Recorded on Days 1, 4 (before and after culling) and on Day 21 of age.
Individual offspring body weights: Days 1, 4, 7, 14, 17, 21 and 25 of age.
Selection of offspring (F1 generation): The selection of offspring to form the F1 generation was made on Day 21 of age.
Selected F1 animals separated from littermates on Day 21 of age.

Postmortem examinations (parental animals):

SACRIFICE
F0 males: After successful littering by females.
F0 females failing to produce a viable litter: Day 25 after mating.
F0 females: Day 21 of lactation.

GROSS NECROPSY
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
The organs weighed and tissue samples fixed are detailed as follows for F0 animals:
- Abnormalities: Organs weighed, and samples fixed.
- Brain: Organs weighed, and samples fixed; From up to three males and three females per group
- Epididymides: Bilateral organs weighed individually; Only one examined - left testis and epididymis reserved for seminology
- Gastrocnemius muscle: Organs weighed, and samples fixed; From up to three males and three females per group
- Ovaries: Organs weighed, and samples fixed.
- Sciatic nerve: Organs weighed and samples fixed; From up to three males and three females per group
- Spinal cord: Organs weighed and samples fixed; From up to three males and three females per group
- Testes: Bilateral organs weighed individually; Only one examined - left testis and epididymis reserved for seminology
- Uterus: Organs weighed, and samples fixed.

Postmortem examinations (offspring):

SACRIFICE
All F1 animals of the selected F1 generation were subject to a detailed necropsy.

Unselected F1 offspring :
Culls - Day 4 of age.
Scheduled kill - Day 21 of age.

Selected F1 offspring: Scheduled kill - Week 8 of age after completion of sexual maturation.


GROSS NECROPSY
After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS
The organs weighed and tissue samples fixed are detailed as follows for F0 animals:
- Abnormalities: Organs weighed, and samples fixed.
- Epididymides: Organs weighed, and samples fixed.
- Testes: Organs weighed, and samples fixed.

This list of tissues preserved was intended to satisfy any possible future requirement for further examination of tissues.

Statistics:

Statistical analyses were performed on the majority of data presented. For some parameters, including estrous cycles and pre coital interval, the similarity of the data was such that analyses were not considered to be necessary. All statistical analyses were carried out separately for males and females. For all adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anomaly and the incidence of the finding within the background control population.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Four premature deaths occurred during the course of the study.
One Control male (No. 3) was despatched for welfare reasons on Day 15 of treatment. This animal showed ante mortem signs of irregular breathing on Day 14 and 15 of treatment and lost 15g body weight overnight. There were no findings at macroscopic examination. As this was a Control animal, this death was not related to Araldite PT 910 administration.
One Control female (No. 41) was found dead shortly after dosing on Day 15 of treatment, before the animals were paired for mating. This animal showed no ante mortem signs. Macroscopic examination revealed dark areas on the lungs, clotted blood in the thoracic cavity, a dark thymus and abnormal fluid in the trachea. As this was a Control animal, this death was not related to Araldite PT 910 administration.
One male that received 3 mg/kg/day (No. 12) was killed for welfare reasons on Day 23 of treatment. This animal had an open wound to its hind limb that was detrimental to its health. The cause of the wound is unknown. There were no other findings at macroscopic examination for this animal.
Another male that received 3 mg/kg/day (No. 13) was killed for welfare reasons on Day 55 of treatment. This animal had lost weight and showed ante mortem signs of decreased activity, cold body temperature, hunched posture and abnormal, yellow skin colour over its whole body. Macroscopic examination revealed yellow adipose tissue, abnormal, firm and reduced contents of the cecum, dark and enlarged kidneys, pale and enlarged liver with an irregular mottled surface, dark areas on the lungs and bronchi and an enlarged spleen with an irregular surface and a dark mass. A relationship to treatment is unlikely as these signs were seen in isolation and with no dose-relationship.
For the remaining F0 animals there were no test item related changes in clinical condition and no signs observed in relation to dose administration.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four premature deaths occurred during the course of the study.
One Control male (No. 3) was despatched for welfare reasons on Day 15 of treatment. This animal showed ante mortem signs of irregular breathing on Day 14 and 15 of treatment and lost 15g body weight overnight. There were no findings at macroscopic examination. As this was a Control animal, this death was not related to Araldite PT 910 administration.
One Control female (No. 41) was found dead shortly after dosing on Day 15 of treatment, before the animals were paired for mating. This animal showed no ante mortem signs. Macroscopic examination revealed dark areas on the lungs, clotted blood in the thoracic cavity, a dark thymus and abnormal fluid in the trachea. As this was a Control animal, this death was not related to Araldite PT 910 administration.
One male that received 3 mg/kg/day (No. 12) was killed for welfare reasons on Day 23 of treatment. This animal had an open wound to its hind limb that was detrimental to its health. The cause of the wound is unknown. There were no other findings at macroscopic examination for this animal.
Another male that received 3 mg/kg/day (No. 13) was killed for welfare reasons on Day 55 of treatment. This animal had lost weight and showed ante mortem signs of decreased activity, cold body temperature, hunched posture and abnormal, yellow skin colour over its whole body. Macroscopic examination revealed yellow adipose tissue, abnormal, firm and reduced contents of the cecum, dark and enlarged kidneys, pale and enlarged liver with an irregular mottled surface, dark areas on the lungs and bronchi and an enlarged spleen with an irregular surface and a dark mass. A relationship to treatment is unlikely as these signs were seen in isolation and with no dose-relationship.
For the remaining F0 animals there were no test item related changes in clinical condition and no signs observed in relation to dose administration.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The body weight gain of males that received 30 mg/kg/day was lower than Control from Day 1 to 22 of treatment. Thereafter body weight gain of these males was comparable to or higher than Control, resulting in the overall body weight gain (Day 1 to 64) being comparable to Control.
The overall body weight gain of males that received 15 or 3 mg/kg/day was slightly lower than Control. Body weight gain between Days 36 and 43 of treatment for males that received 3 mg/kg/day was significantly lower than Control (27% of Control), mainly because two males lost weight during this period, one of which was male number 13 which continued to lose weight and was killed for welfare reasons on Day 55.
Mean body weight gain of females prior to pairing that received 15 or 30 mg/kg/day was comparable to Control. Overall mean body weight gain prior to pairing for females that received 3 mg/kg/day was slightly lower than Control. For females that received 30 mg/kg/day, overall mean body weight gain during gestation (Day 0 to 20) was lower than Control due to low weight gain (64% of Control) between Days 14 and 20 of gestation. Overall mean body weight gain for females that received 15 or 3 mg/kg/day was higher than Control during gestation.
Overall mean body weight gain during lactation (Day 1 to 21) was slightly lower than Control for all groups of treated females, however no dose-relationship was apparent.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

There was a dose-related decrease in progressively motile sperm with a significant decrease from Control observed in males that received 30 mg/kg/day. Some associated sperm motion parameters; VAP (average path velocity), VSL (progressive or straight-line velocity) and VCL (curvilinear velocity or track speed) showed a decrease, compared to Controls, in males that received 30 mg/kg/day and a dose-related decrease was observed for VAP and VCL. This may indicate a slight decrease in the sperms ability to perform in the forward motion. In the absence of any morphological effects, this could be due to epididymal dysfunction or a possible depletion of energy stores.
In males that received 30 or 15 mg/kg/day there was a decrease in cauda epididymal sperm numbers compared to Control, with the lowest count evident in males that received Araldite PT 910 at 30 mg/kg/day. There was no effect of treatment on cauda epididymal sperm numbers in males that received 3 mg/kg/day. There were also statistically significant decreases in testicular spermatid numbers and concentration in all groups of treated males compared to Control, with the lowest count/concentration observed in males that received 3 mg/kg/day.

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

All females that received Araldite PT 910 at 30 mg/kg/day had a gestation length of 23 days. Although a gestation of this length is normal, it is not common for all females within a group to have a gestation of this length, however this is likely to be due to the smaller litter size in this dose group and therefore less pressure on the females to give birth.
The gestation length and index of animals that received 15 or 3 mg/kg/day were unaffected by treatment.

Effect levels (P0)

Dose descriptor:

NOAEC

Effect level:

3 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

reproductive function (sperm measures)

reproductive performance

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

There was no clear effect of treatment on the reproductive organ weights of male offspring, however, when compared to Control, the absolute and body weight adjusted testes weights of male offspring that received 30 mg/kg/day were marginally higher than Control.

Gross pathological findings:

no effects observed

Effect levels (F1)

Dose descriptor:

NOAEC

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects noted in F1 generation

Overall reproductive toxicity

Reproductive effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results obtained in this preliminary study of reproductive performance, it was considered that oral gavage administration of Araldite PT 910 to Crl:CD(SD) rats at 30 mg/kg/day is not suitable for investigation in a main extended one-generation reproductive toxicity study because of the adverse effects on reproductive performance. It is therefore concluded that a dose level of 15 mg/kg/day would be a suitable high dose for investigation in a main study. Although there were apparent slight reductions in testicular and epididymal spermatid numbers in animals that received Araldite PT 910 at 15 mg/kg/day, reproductive performance was good and there was no effect of treatment on litter size, sex ratio or offspring survival and direct treatment of the selected F1 generation from Day 21 of age was well tolerated at all dose levels investigated.

Executive summary:

 Summary

The purpose of this study was to assess the influence of Araldite PT 910, an industrial chemical, on reproductive performance when administered orally (gavage) to CD rats, and to establish suitable dose levels for an extended one-generation reproductive performance study.

For the F0 generation, three groups of eight male and eight female rats receivedAraldite PT 910at doses of 3, 15 or 30 mg/kg/day by oral gavage administration for two weeks before pairing until termination. A similarly constituted Control group received the vehicle, propylene glycol, at the same volume dose as treated groups. The F1 generation comprised of ten male and ten female progeny from Group 1-3 andfive male and five female progeny from Group 4. They received direct treatment withAraldite PT 910from Day 21 of age until termination after attainment of sexual maturity. 

During the study, clinical condition, body weight, food consumption, estrous cycles, pre‑coital interval, mating performance, fertility, gestation length, seminology, organ weight and macroscopic pathology investigations were undertaken on the F0 generation. The clinical condition of offspring, litter size and survival, sex ratio, sexual maturation (selected F1 generation only) and body weight was assessed and organ weight (males only) and macroscopic pathology investigations were undertaken on the F1 generation.

Results

F0 – F1 generation

Four premature deaths occurred in the F0 generation during the course of the study, including two in Controls; none were considered to be related to Araldite PT 910 administration.

For the remaining F0 animals there were no test item related changes in clinical condition and no signs observed in relation to dose administration.

The body weight gain of males that received 30 mg/kg/day was lower than Control from Day 1 to 22 of treatment. Thereafter body weight gain of these males was comparable to or higher than Control, resulting in the overall body weight gain (Day 1 to 64) being comparable to Control.

The overall body weight gain of males that received 15 or 3 mg/kg/day was slightly lower than Control. Body weight gain between Days 36 and 43 of treatment for males that received 3 mg/kg/day was significantly lower than Control (27% of Control), mainly because two males lost weight during this period, one of which was male number 13 which continued to lose weight and was killed for welfare reasons on Day 55.

Mean body weight gain of females prior to pairing that received 15 or 30 mg/kg/day was comparable to Control. Overall mean body weight gain prior to pairing for females that received 3 mg/kg/day was slightly lower than Control. For females that received 30 mg/kg/day, overall mean body weight gain during gestation (Day 0 to 20) was lower than Control due to low weight gain (64% of Control) between Days 14 and 20 of gestation. Overall mean body weight gain for females that received 15 or 3 mg/kg/day was higher than Control during gestation.

Overall mean body weight gain during lactation (Day 1 to 21) was slightly lower than Control for all groups of treated females, however no dose-relationship was apparent.

During the first two weeks of treatment prior to pairing for males and females, food intake was unaffected by treatment. Throughout gestation, the food intake of all groups of treated females was slightly higher than Control. During lactation, the food intake of females that received 30 mg/kg/day was lower than Control from Day 7 to 21 of lactation. The food intake during lactation of females that received 15 or 3 mg/kg/day was comparable to Control.

There was no effect of treatment on estrous cycle regularity or pre-coital interval at any dose level investigated.

Mating performance, as assessed by percentage mating was unaffected by treatment, however the conception rate and fertility index were lower than Control for animals that received Araldite PT 910 at 15 mg/kg/day and markedly lower than Control for animals that received 30 mg/kg/day (88% and 63% respectively). There was no effect of treatment on mating performance or fertility for animals that received 3 mg/kg/day.

All females that received Araldite PT 910 at 30 mg/kg/day had a gestation length of 23 days. Although a gestation of this length is normal, it is not common for all females within a group to have a gestation of this length, however this is likely to be due to the smaller litter size in this dose group and therefore less pressure on the females to give birth. The gestation length and index of animals that received 15 or 3 mg/kg/day were unaffected by treatment.

There was a dose-related decrease in progressively motile sperm with a significant decrease from Control observed in males that received 30 mg/kg/day. Some associated sperm motion parameters; VAP (average path velocity), VSL (progressive or straight-line velocity) and VCL (curvilinear velocity or track speed) showed a decrease, compared to Controls, in males that received 30 mg/kg/day and a dose-related decrease was observed for VAP and VCL. This may indicate a slight decrease in the sperms ability to perform in the forward motion. In the absence of any morphological effects, this could be due to epididymal dysfunction or a possible depletion of energy stores. 

In males that received 15 or 30 mg/kg/day there was a decrease in cauda epididymal sperm numbers compared to Control, with the lowest count evident in males that received Araldite PT 910 at 30 mg/kg/day. There was no effect of treatment on cauda epididymal sperm numbers in males that received 3 mg/kg/day. There were also statistically significant decreases in testicular spermatid numbers and concentration in all groups of treated males compared to Control, with the lowest count/concentration observed in males that received 3 mg/kg/day so a relationship to treatment is uncertain.

Absolute and body weight adjusted testes and epididymal weights were marginally lower than Control in males that received Araldite PT 910 at 30 mg/kg/day. There was no effect of treatment on male organ weights at 15 or 3 mg/kg/day. There was no effect of treatment on female brain or ovarian weights. The absolute and body weight adjusted mean uterus weights for females that received 30 mg/kg/day were lower than Control and other treated groups.

Macroscopic findings at scheduled termination were limited to pale areas on the lungs and bronchi for one female that received 30 mg/kg/day, hair loss in one female that received 30 mg/kg/day and one female that received 15 mg/kg/day and a skin depression and scabs on one female that received 15 mg/kg/day.

Four females that received 30 mg/kg/day and one female that received 15 mg/kg/day failed to litter. Of these, three of the females that received 30 mg/kg/day and the female that received 15 mg/kg/day were confirmed not to be pregnant at necropsy with no sign of any implantations. One female that received 30 mg/kg/day was confirmed to have been pregnant with two implantations but at macroscopic examination was found with one autolyzed dead fetus in the left horn and one early resorption in the right horn.

Amongst the offspring, there were no signs at physical examination related to treatment.

Females that received Araldite PT 910 at 30 mg/kg/day had a significantly lower mean number of implantations compared to Control. As a result, mean litter sizes in this dose group were also significantly lower than Control. In this high dose group, offspring post implantation, live birth, viability and lactation survival indices and sex ratio were unaffected by parental treatment. For animals that received 15 or 3 mg/kg/day, litter size, offspring survival and sex ratio were unaffected by parental treatment.

Group mean body weights of male and female offspring derived from parents that received 30 mg/kg/day were slightly higher than Control, probably reflecting the lower litter size. This difference was significant between Days 1 and 7 of ageand while weight gain from Days 1 to 4 of age was higher than Control, overall weight gain to weaning was similar to Control. Overall group mean body weight gain from Day 1 to 21 of age of male and female offspring derived from parents that received 15 or 3 mg/kg/day were slightly lower than Control.

Amongst the unselected offspring, there were no findings observed at macroscopic examination.

Selected F1 generation

Amongst the selected F1 offspring, there were no test item related changes in clinical condition and no signs observed in relation to dose administration.

Overall mean body weight gain of male offspring was unaffected by treatment.

Overall mean body weight gain of female offspring treated at 30 mg/kg/day was slightly higher than Control, probably reflecting their slightly greater body weights at the start of the F1 generation. Mean body weight gain of female offspring treated at 15 or 3 mg/kg/day was comparable to Control throughout the treatment period.

The food intake of female offspring that received 30 mg/kg/day was slightly higher than Control from Day 8 after the nominal start date. The food intake of male offspring that received 30 mg/kg/day and male and female offspring that received 15 or 3 mg/kg/day was unaffected by Araldite PT 910 administration.

There was no effect of treatment on balano preputial separation or vaginal opening when compared to Controls.

There was no clear effect of treatment on the reproductive organ weights of male offspring, however, when compared to Control, the absolute and body weight adjusted testes weights of male offspring that received 30 mg/kg/day were marginally higher than Control.

There were no macroscopic abnormalities detected at scheduled termination of the selected F1 offspring, therefore there are no data for this parameter present in the report.

Conclusion

Based on the results obtained in this preliminary study of reproductive performance, it was considered that oral gavage administration of Araldite PT 910 to Crl:CD(SD) rats at 30 mg/kg/day is not suitable for investigation in a main extended one-generation reproductive toxicity study because of the adverse effects on reproductive performance.

It is therefore concluded that a dose level of 15 mg/kg/day would be a suitable high dose for investigation in a main study. Although there were apparent slight reductions in testicular and epididymal spermatid numbers in animals that received Araldite PT 910 at 15 mg/kg/day, reproductive performance was good and there was no effect of treatment on litter size, sex ratio or offspring survival and direct treatment of the selected F1 generation from Day 21 of age was well tolerated at all dose levels investigated.