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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

NOAEL applied based on the results of a two week prelimininary range finding study.  In the Main EOGRTs study, no similar effects were observed.

Preliminary EOGRTs Study, (non guideline)

A preliminary, dose range finding study was conducted using three groups of eight male and eight female rats and dose levels of 3, 15 or 30 mg/kg bw/d. The rats were dosed via oral gavage for two weeks before pairing.

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

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 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 at 30 mg/kg/day.

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.

A dose level of 30 mg/kg bw/d was deemed not suitable for investigation in the main Extended One Generation Study owing to the effects on reproductive performance. Despite slight reductions in testicular and epididymal spermatic numbers, 15 mg/kg bw/d was deemed suitable for the longer treatment duration neccessary for the main Extended One Generation study.

Main Extended One Generation Study (OECD 443)

In theF0generation, three groups of 24 male and 24 female rats received the test item at dose levels of 2.5, 6 or 15 mg/kg/day at a volume dose of 2.5 mL/kg/day. Males were treated for ten weeks before pairing, up to necropsy after litters were weaned. Females were treated for ten weeks before pairing, throughout pairing up to necropsy on Day 28 of lactation. In theF1generation, 60 males and 60 females were treated from weaning to their scheduled termination (relevant to each cohort) at the same dose levels and volume-dose as theF0generation. A similarly constituted Control group received the vehicle, propylene glycol at the same volume dose. In addition, for the F0 generation only, one group of 24 males were treated at 15 mg/kg/day for two weeks before pairing with 24 untreated females who reared their litters to weaning, this additional group was included to mirror the test designs used in the two preliminary range finding tests.  

Litter size, offspring survival, sex ratio, offspring body weight gain and ano-genital distance were unaffected at all dose levels investigated.

There was no effect of treatment at any dose level investigated on testosterone or luteinising hormone concentrations among F0 animals, or on serum T4 or TSH concentrations in F0 or F1 Cohort 1A adult animals, or in the F1 offspring on Day 22 of age.

In F0 and F1 Cohort 1A males some slight, non-dose-dependent decreases in sperm motion parameters were apparent, at 6 or 15 mg/kg/day among F0 males and at 15 mg/kg/day in F1 Cohort 1A males. In the absence of any histopathological changes in the reproductive tract of F0 or F1 Cohort 1A males, or any effects on mating performance or fertility of the F0 males, these minor changes in the sperm motion parameters were considered to be of no toxicological significance within the context of this study. 

In the F0 generation, there was no evidence of discernible difference in the mating performance/fertility or detailed sperm analysis of males given 15 mg/kg/day for two weeks prior to pairing (approximately 10 weeks of treatment in total) compared with the males given 15 mg/kg/day for 10 weeks prior to pairing (approximately 18 weeks of treatment in total). The lack of discernible difference in the sperm analysis of the two-week dosing contradicts the findings within the preliminary range finding studies. This may indicate that effects on sperm within the first two weeks of dosing are a transient phenomenon associated with adaptation to early toxicological conflict or dosing procedures.

Based on the results obtained in this study it was concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity among F0 and F1 adult animals, the reproductive performance of the F0 generation animals, F1 offspring growth and survival to weaning, and developmental neurobehaviour and neuropathology of the F1 animals was 15 mg/kg/day. 

It was also concluded that there was no adverse effect on the number or quality of the sperm of F0 or F1 Cohort 1A males, and in the F0 generation there was no discernibledifference in the mating performance/fertility or detailed sperm analysis of males given 15 mg/kg/day for two weeks prior to pairing (approximately 10 weeks of treatment in total) compared with the males given 15 mg/kg/day for 10 weeks prior to pairing (approximately 18 weeks of treatment in total).

Link to relevant study records

Reference

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.

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.

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.

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

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

Dose / conc.:

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

Remarks:

F0 and F1 generation

Dose / conc.:

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

Remarks:

F0 and F1 generation

Dose / conc.:

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

Remarks:

F0 and F1 generation

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.

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.

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: 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.

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

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

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

Reproductive effects observed:

yes

Lowest effective dose / conc.:

15 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to other toxic effects:

not specified

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

NOAEL applied based on the results of a two week prelimininary range finding study. Main EOGRTs study in progress, due to effects on male fertility a precautionary NOAEL has been applied until the final study results are available.

Effects on developmental toxicity

Description of key information

Embryo-fetal survival was considered unaffected by treatment in an OECD 414 study with the test item, but there were a number of findings at fetal pathology examination indicative of fetuses being at a transient stage in fetal development and not considered adverse. Therefore, the No-Observed-Adverse- Effect-Level (NOAEL) for maternal toxicity and for embryo-fetal toxicity was concluded to be 60 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 October 2017 - 14 March 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Reputable global supplier
- Age at study initiation: Approximately 72 days old.
- Weight at study initiation: 229 to 293 g.


- Fasting period before study: none
- Housing: Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet (e.g. ad libitum): SDS VRF1 Certified pelleted diet. Non-restricted.
- Water (e.g. ad libitum): Non-restricted.
- Acclimation period: Six days before commencement of pairing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- 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.

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 weighings of the test item.

VEHICLE - Propylene glycol
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: n/a
- Amount of vehicle (if gavage): 2.5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, limit of detection and quantification, linearity of detector response, repeatability, method accuracy and precision.
The homogeneity and stability was confirmed for Araldite PT 910 in propylene glycol formulations at nominal concentrations of 1 mg/mL and 400 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, ambient temperature storage (15 to 25ºC) for 1 day and refrigerated storage (2 to 8ºC) for up to 15 days.
The mean concentrations of Araldite PT 910 in test formulations analyzed for the study were within +10/-15% of nominal concentrations, confirming accurate formulation. The difference between the samples remained within 2%, confirming precise analysis.

Details on mating procedure:

- Impregnation procedure: cohoused

- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.
A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

19 days

Frequency of treatment:

once daily

Duration of test:

19 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Control

Dose / conc.:

20 mg/kg bw/day

Remarks:

Group 2

Dose / conc.:

60 mg/kg bw/day

Remarks:

Group 3

Dose / conc.:

200 mg/kg bw/day

Remarks:

Group 4

No. of animals per sex per dose:

20 females

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on the effects of a preliminary embryo-fetal study at this laboratory (Envigo study number WB30PP) which investigated dose levels of 50, 100 or 200 mg/kg/day.
- Rationale for animal assignment (if not random): random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0-20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0, 1-2, 3-5, 6-9, 10-13, 14-17 and 18-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals were killed on Day 20 after mating
- All adult animals were subject to a detailed 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.

OTHER:
Reproductive Assessment
The following were recorded for all animals:
Uterus - Gravid uterine weight (including cervix and ovaries).
For each ovary/uterine horn - Number of: Corpora lutea, Implantation sites, Resorption sites (classified as early or late), and Fetuses (live and dead).
Apparently non pregnant animals - The number of uterine implantation sites were checked after staining with ammonium sulphide.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses, live and dead

Fetal examinations:

- External examinations: Yes - all viable fetuses and placentae
- Soft tissue examinations: Yes - all viable fetuses and placentae
- Skeletal examinations: Yes - all viable fetuses and placentae
- Head examinations: No

Statistics:

- Bartlett's test
- Williams’ test
- Dunnett's test
- Kruskal-Wallis’ test
- Wilcoxon rank sum tests
- Shirley's test
- Steel's test
- Fisher’s exact tests

Indices:

Litter size and survival indices.

Historical control data:

Available from this laboratory

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test-material related macroscopic abnormalities detected in the adult females at scheduled termination on Day 20 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the numbers of implantations, resorptions (classified as early or late), live young, sex ratio or pre- and post-implantation loss.

Details on results:

There were no deaths on this study.
There were no signs seen at physical examinations or at post-dose observation considered related to treatment with Araldite PT 910.

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:

not examined

Details on maternal toxic effects:

There was no effect of treatment on the numbers of implantations, resorptions (classified as early or late), live young, sex ratio or pre- and post-implantation loss.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

changes in number of pregnant

changes in pregnancy duration

clinical signs

dead fetuses

early or late resorptions

effects on pregnancy duration

food consumption and compound intake

gross pathology

mortality

number of abortions

pre and post implantation loss

total litter losses by resorption

Key result

Abnormalities:

no effects observed

Localisation:

not specified

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

For females which received Araldite PT-910 at 200 mg/kg/day, litter weight and fetal weight (male, female and overall) was statistically significantly lower than that of the control as well as if compared to those animals which received 20 and 60 mg/kg/day.
There was no effect of treatment on placental, litter or fetal weights in animals which received Araldite PT-910 at 20 or 60 mg/kg/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

For females which received Araldite PT-910 at 200 mg/kg/day, litter weight and fetal weight (male, female and overall) was statistically significantly lower than that of the control as well as if compared to those animals which received 20 and 60 mg/kg/day.
There was no effect of treatment on placental, litter or fetal weights in animals which received Araldite PT-910 at 20 or 60 mg/kg/day.

Changes in postnatal survival:

not specified

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Also, at 200 mg/kg/day there was an increased incidence of variation in lens shape and delayed/incompletely ossified/unossified sacrocaudal vertebral arches compared to concurrent control and outside of HCD. These findings are indicative of a delay in fetal development and may be attributed to the very slight decrease in mean fetal bodyweight seen at this dose level. Both abnormalities are a transient stage in fetal development and would therefore not be considered adverse.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 200 mg/kg/day there were 3 fetuses/litters with bent scapula(e), short/thickened humerus with associated medially thickened/kinked/incompletely ossified ribs which were just outside of litter Historical Control Data (HCD). This is a known combination of abnormalities sometimes seen in the Crl: CD(SD) strain, including control groups. At such small values this is not thought to be treatment related.

Visceral malformations:

not examined

Other effects:

not examined

Details on embryotoxic / teratogenic effects:

Mean fetal weights were slightly reduced. Embryo-fetal survival was considered unaffected by treatment, but there were a number of findings at fetal pathology examination indicative of fetuses being at a transient stage in fetal development and not considered adverse.
Therefore, the No-Observed- Effect-Level (NOEL) for maternal toxicity and for embryo-fetal toxicity was concluded to be 60 mg/kg/day.

Key result

Dose descriptor:

NOEL

Effect level:

ca. 60 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

changes in sex ratio

fetal/pup body weight changes

changes in litter size and weights

external malformations

skeletal malformations

Key result

Abnormalities:

effects observed, non-treatment-related

Localisation:

skeletal: scapule

skeletal: rib

skeletal: vertebra

Description (incidence and severity):

There were a number of findings at fetal pathology examination in the 200 mg/kg/day group, including an increased incidence of variation in lens shape and delayed/incompletely ossified/unossified sacrocaudal vertebral arches compared to concurrent control and outside of HCD. These minor findings are indicative of a delay in fetal development and may be attributed to the very slight decrease in mean fetal bodyweight seen at this dose level. Both abnormalities are a transient stage in fetal development and would therefore not be considered adverse.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

Treatment related:

no

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

not specified

Relevant for humans:

yes

Results from preliminary study - Araldite PT 910: Preliminary Study for Effects on Embryo-Fetal Development in the Rat by Oral Gavage Administration (Envigo ref WB30PP)

Maternal Responses

There were no unscheduled deaths, clinical signs or signs related to dosing that were considered related to treatment with Araldite PT 910.

Body Weight and Gravid Uterine Weight

Females receiving Araldite PT 910 at 200 mg/kg/day showed body weight stasis from the start of treatment (Day 1 of gestation) until Day 3 of treatment (Day 4 of gestation), compared with body weight gain of 8g in the Control group. From Day 4-18 of gestation body weight gain of these females was similar to Control. From Day 18 of gestation, body weight gain of females receiving 200 mg/kg/day was lower than Control resulting in the overall body weight gain (Days 1-20 of gestation) of these females being lower than Control (79% of Control). The body weight change for these females prior to treatment (Day 0-1 of gestation) was also lower than Control. 

Body weight gain of females receiving Araldite PT 910 at 100 mg/kg/day was similar to Control from the start of treatment until Day 18 of gestation. From Day 18 of gestation body weight gain of these females was lower than Control resulting in the overall body weight gain for these females being slightly lower than Control (92% of Control).

There was no effect of treatment on body weight gain for females which received Araldite PT 910 at 50 mg/kg/day.

The gravid uterus weight and maternal weight gain adjusted for the uterine weight was lower than Control for females receiving 200 mg/kg/day.

There was no effect of treatment on the gravid uterus weight or maternal weight change adjusted for the uterine weight of females which received Araldite PT 910 at 100 or 50 mg/kg/day.

Food Consumption

Following the start of study (Day 0 of gestation) and throughout gestation, food consumption of females receiving Araldite PT 910 at 200 mg/kg/day was slightly lower than Control and when compared with the pre dose intake. The food consumption of females receiving Araldite PT 910 at 100 or 50 mg/kg/day was similar to Control throughout the study.

Macropathology

Macroscopic findings in the adult females and fetuses at necropsy on Day 20 of gestation were limited to one female in the 200 mg/kg/day group with dilated pelvis; kidney.

Reproductive Assessment

The mean corpora lutea count was lower in the treated groups compared to Control. This is not treatment related because corpora lutea formed before treatment commenced. The resultant mean number of implantations was also lower in the treated groups compared to Control and given that there was no effect of treatment on pre-implantation loss, this is likely a result of a higher corpora lutea count in controls and not an effect of treatment. There was no effect of treatment on the number of early or late resorptions or sex ratio. 

Post-implantation loss was slightly higher than Control in females receiving Araldite PT 910 at 200 or 100 mg/kg/day and the resultant mean number of live young was also lower than Control in these groups.

In females receiving Araldite PT 910 at 50 mg/kg/day the mean number of live young was lower than Control, but given that there was no effect of treatment on pre- or post‑implantation loss in this group, this is a result of a higher corpora lutea and resulting higher implantation count and number of live young in the controls.

Placental, Litter and Fetal Weights

There was no effect of treatment on placental weight.  

Amongst the litters of females receiving Araldite PT 910 at 200 mg/kg/day mean fetal and litter weights were slightly lower than Control. There was no effect of treatment on fetal or litter weight in the litters of females receiving Araldite PT 910 at 100 or 50 mg/kg/day.

Conclusion

Based on these results, the high dose level for the main OECD 414 study could be 200 mg/kg/day, aiming to induce some signs of toxicity. Possible low and intermediate dose levels could be 20 and 60 mg/kg/day to investigate the dose response of any potential toxicity observed.

Conclusions:

In this study, treatment with Araldite PT-910 at 20 or 60 mg/kg/day was generally well tolerated.
At 200 mg/kg/day, maternal body weight performance and food consumption was lower than that of the Control after the start of treatment (early gestation) and again between Days 18-20 of gestation. The gravid uterine weight and bodyweight gain when adjusted for the weight of the gravid uterus of females which received Araldite PT-910 at 200 mg/kg/day was lower than that of the control. Mean fetal weights were slightly reduced. Embryo-fetal survival was considered unaffected by treatment, but there were a number of findings at fetal pathology examination indicative of fetuses being at a transient stage in fetal development and not considered adverse.
Therefore, the No-Observed- Effect-Level (NOEL) for maternal toxicity and for embryo-fetal toxicity was concluded to be 60 mg/kg/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Toxicity to reproduction: other studies

Description of key information

A non-guideline, investigatative study was conducted to further understand the potential for recovery of effects noted on the preliminary, range finding Extended One Generation study. Two groups, each comprising eight male Crl:CD(SD) rats, received Araldite PT 910 at doses of 15 or 30 mg/kg/day. A similarly constituted control group received the vehicle, propylene glycol, at the same volume dose as treated groups. A further 24 male rats (eight males per recovery period) were assigned to each of the control and treated groups. These animals were treated for 14 days, followed by a one week, two weeks or four weeks period without treatment to assess the potential for any treatment-related change to recover.

Minimal effects of treatment were seen on sperm motility after treatment for 15 days at 30 and 15 mg/kg/day, there was an increase in cauda epididymal concentration and numbers (numbers not increased at 15 mg/kg/day) and a decrease in testicular spermatid concentration (statistically significant at 30 mg/kg/day) and numbers, observed across all treated groups compared to concurrent control. Gradual recovery was observed on Day 8 and Day 15 of recovery, with complete recovery by Day 29 of recovery. In conclusion, complete sperm recovery did occur in male rats by Day 29 of recovery.

Full study summary can be found in Toxicity to Reproduction.

Justification for classification or non-classification

A classification of Reproductive toxicity Category 2, H361: Suspected of damaging fertility or the unborn child has been selected owing to the effects on sperm noted in preliminary Extended One Generation studies, where animals recieved 2 weeks treatment pre-necropsy. The 2 week pre-pairing effects were shown to be reversible after 29 days post-treatment.

No similar, significant, effects were seen on the main study, where male animals were dosed for two weeks pre-paring (total of 10 weeks dosing) or with 10 weeks pre-pairing (18 weeks total treatment).

The lack of discernible difference in the sperm analysis conducted with 2 weeks prepairing, 10 week treatment, from control animal data contradicts the findings seen on the preliminary, range finding studies. This may indicate that the effects seen on sperm within the first two weeks of dosing are a transient phenomenon associated with adaptation to early toxicological conflict or dosing procedures.