Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The higest dose set for the reproductive/developmental screening study was 100 mg/kg/day this was also defined as the No-observed-adverse-effect-level (NOAEL) for reproductive performance which was also 100 mg/kg/day.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 February 2017 -

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Revised 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

other: Crl:CD(SD) rat.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males 70 to 77 days old and Females 83 to 90 days old.
- Weight at study initiation: Males: 311-386 g; Females: 220-269 g
- Fasting period before study: No
- Housing:Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing (treatment), gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing.
- Diet (e.g. ad libitum):Non-restricted.
- Water (e.g. ad libitum):Non-restricted.
- Acclimation period:Males: Six days before commencement of treatment. Females: 20 days before commencement of treatment.
ENVIRONMENTAL CONDITIONS
- Temperature (°C):Monitored and maintained within the range of 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.
IN-LIFE DATES: From: 15 February 2017 To: 29 April 2017

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):Weekly.
- Storage temperature of food:Refrigerated (2 to 8°C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Not specified.
- Concentration in vehicle: The required amount of test item was weighed and approximately 50% of the final volume of vehicle was added to the test item.

Details on mating procedure:

- M/F ratio per cage:1:1 from within the same treatment groups.
- Length of cohabitation: Up to two weeks.
- Proof of pregnancy:Ejected copulation plugs in cage tray and sperm in the vaginal smear. When positive evidence of mating was detected this was considered as Day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males were treated daily for 15 days before pairing, up to necropsy after a minimum of four consecutive weeks.
Females were treated daily for 15 days before pairing, throughout pairing, gestation and until Day 12 of lactation.
The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

50 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 animals per sex per dose.

Details on study design:

- Dose selection rationale: The dose levels for this study were set following a review of the results from the 28 day oral toxicity study conducted with AO-26 (Huntingdon Life Sciences Study Number CVJ0157). In that study a NOAEL (No observed adverse effect level) could not be established from dose levels of 100, 300 or 1000 mg/kg/day due to adverse findings from pathological investigations revealing treatment-related changes in the liver [periportal hepatocellular vacuolation] and heart [inflammatory cell infiltrate associated with diffuse myocardial degneration (vacuolation)]. Following consultation with the Sponsor the high dose was limited to 100 mg/kg/day because of the pathological heart changes that were evident on the OECD 407 study and the theoretical potential to adversely affect animals during mating, parturition and lactation. The low and intermediate dose levels (25 and 50 mg/kg/day) were selected with the aim to provide a no observed adverse effect level for reproductive/developmental toxicity. Secondary to the study objective these dose levels also provide additional pathology information on the target organs at dose levels <100 mg/kg/day.
- Rationale for animal assignment (if not random):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.
The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 males
Once each week

F0 females
Once each week until Pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 6 and 13

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males
Before dosing on the day that treatment commenced (Week 0) and weekly thereafter. On the day of necropsy.

F0 females
Before dosing on the day that treatment commenced (Week 0) and weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7 and 13 of lactation. On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded as follows:

F0 males
Weekly before pairing, from the day that treatment commenced. For females after mating food consumption was performed to match the body weight recording:
Days 0-6, 7-13 and 14-19 after mating
Days 1-3, 4-6 and 7-12 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

Oestrous cyclicity (parental animals):

Dry and wet smears were taken as follows:
Dry smears: For 15 days before pairing using cotton swabs.
Wet smears: Using pipette lavage during the following phases:
-For 14 days before treatment (all females including spares)
-After pairing until mating.
-For four days before scheduled termination

Litter observations:

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 1-13 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 13 of age.
Individual offspring body weights: Days 1, 4, 7 and 13 of age.
Ano-genital distance: Day 1 - all F1 offspring.
Nipple/areolae count: Day 13 of age - male offspring.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: After 4 weeks of treatment.
- Maternal animals: Day 13 of lactation.

GROSS NECROPSY
- 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.

Epididymides (caput, corpus and cauda)
Heart
Kidney
Liver
Ovaries
Pituitary
Prostate
Seminal vesicles (with coagulation gland)
Testes
Thyroids
Uterus with cervix and oviducts
Vagina

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at [13] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Where possible, a fresh macroscopic examination (external) with an assessment of stomach for milk content was performed.An externally macroscopic examination; particular attention was paid to the eternal genitalia.All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Animals observed with external abnormalities
were retained pending possible future examination. Thyroid glands were preserved from one male and one female in each litter.

GROSS NECROPSY
- Gross necropsy as above for parental animals.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre-coital interval, mating performance, gestation index and stage of estrous cycle at termination 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 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.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Food consumption, over appropriate study periods during gestation and lactation
Estrous cycles during treatment
Gestation length
Litter size, survival indices and sex ratio
Ano-genital distance, adjusted for Day 1 body weight
Organ weights, both absolute and adjusted for terminal body weight

The following comparisons were performed:

Group 1 vs 2, 3 and 4

Reproductive indices:

For litter size and survival indices, if 75% of the data (across all groups) were the same value, for example c, Fisher’s exact tests (Fisher 1973) were performed. Treatment groups were compared using pairwise comparisons of each dose group against the control both for i) values c, as applicable.

Clinical signs:

no effects observed

Description (incidence and severity):

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to treatment.

Dermal irritation (if dermal study):

not examined

Mortality:

not specified

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight gain for males receiving AO-26 during Week 1 of treatment was slightly but significantly high when compared with Controls (p<0.05); there was no evidence of a dose response. Thereafter, mean bodyweight change for males receiving AO-26 at all dose levels were similar to Controls.
The body weight gain for females before pairing for mating was unaffected by treatment. During Days 0 to 7 of gestation females receiving 100 mg/kg/day showed slightly low weight gain, approximately 83% of Controls and overall weight gain at this dose level was approximately 90% of Controls; these differences did not attain statistical significance. At 25 and 50 mg/kg/day the body weight gain of females during gestation was essentially similar to Controls.
Body weight gain during lactation was unaffected by treatment with AO-26.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males and females for the two weeks before pairing and for females
during gestation and lactation was unaffected by treatment.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Changes related to treatment with AO-26 were seen in the heart.
Increased incidence and severity of myocardial degeneration associated with or without increased incidence and severity of inflammatory cell infiltrate were seen in some males that received 25 mg/kg/day and all males that received 50 or 100 mg/kg/day with a clear doserelationship. In the females, dose- related myocardial degeneration was seen in some animals that received 25, 50 or 100 mg/kg/day and was accompanied by inflammatory cell infiltrate in one female that received 50 mg/kg/day and some females that received 100 mg/kg/day.
The majority of the degenerate myofibres displayed some level of vacuolation.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There was no effect of treatment on estrous cycles, pre-coital interval or mating performance and fertility. Gestation length and gestation length were also unaffected by treatment. At termination all lactating females were in diestrus.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Description (incidence and severity):

There was no effect of treatment on estrous cycles, pre-coital interval or mating performance and fertility. Gestation length and gestation length were also unaffected by treatment. At termination all lactating females were in diestrus.

Treatment of AO-26 to parental animals at 25, 50 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility or macropathology.

Adverse findings were limited to an increase in heart weight for males that received 50 or 100 mg/kg/day and this correlated with histopathology which showed myocardial degeneration associated with or without inflammatory cell infiltrate which was seen in the heart of both male and females that received 25, 50 or 100 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

25 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that could be attributed to parental treatment.

Dermal irritation (if dermal study):

not examined

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight of male and female offspring on Day 1 of age and subsequent weight gain up to Day 13 of age was unaffected by treatment.

Sexual maturation:

no effects observed

Description (incidence and severity):

On Day 1 of age the mean absolute and body weight adjusted ano-genital distance for both male and female offspring derived from the groups receiving AO-26 were similar to Controls and considered to be unaffected by parental treatment and are therefore considered to be incidental.

Other effects:

no effects observed

Description (incidence and severity):

Offspring Nipple Count

There was a low incidence of males observed with nipples on Day 13 of age in one litter at 25 mg/kg/day, one litter at 50 mg/kg/day and three litters at 100 mg/kg/day. In the absence of any effect on ano-genital distance it is unlikely that these findings are associated with treatment.

Offspring Macropathology

Macroscopic examination of offspring that died before scheduled termination and those that were killed at scheduled termination on Day 13 of age did no treveal any findings that could be attributed to parental treatment with AO-26.

The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

sexual maturation

clinical signs

mortality

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Treatment of AO-26 to parental animals at 25, 50 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility or macropathology. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment. Adverse findings were limited to an increase in heart weight for males that received 50 or 100 mg/kg/day and this correlated with histopathology which showed myocardial degeneration associated with or without inflammatory cell infiltrate which was seen in the heart of both male and females that received 25, 50 or 100 mg/kg/day. It was therefore concluded that the No-observed-adverse-effect-level (NOAEL) for reproductive performance was 100 mg/kg/day; however a NOAEL for parental toxicity was not established.

Executive summary:

The purpose of this study was a screening test for reproductive/development effects, with administration of the test item AO-26, an antioxidant for plastics, by oral gavage administration for at least four weeks.

Three groups of ten male and ten female rats received AO-26 at doses of 25, 50 or 100 mg/kg/day by oral gavage administration. Males were treated daily for 15 days before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for 15 days before pairing, throughout pairing, gestation and until Day 12 of lactation.

Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was inutero or via the milk. A similarly constituted Control group received the vehicle, corn oil, at the same volume dose as treated groups. During the study, clinical condition, body weight, food consumption, estrous cycles, precoital

interval, mating performance, fertility, gestation length, organ weight and macroscopic pathology and histopathology investigations were undertaken. The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance and nipple counts (males only), and macropathology for all offspring were also recorded. In addition Thyroxine levels were assessed for offspring on Day 13 of age and for adult males at scheduled termination.

Results

Analyses of samples for thyroxine (T4) obtained from adult male animals after four weeks of treatment and F1 offspring on Day 13 of age did not reveal any differences that could be attributed to treatment; therefore further assessment of T4 and thyroid stimulating hormone (TSH) was not considered necessary.

F0 responses

No signs were observed in association with dose administration and there were no signs at routine physical examination that could be attributed to treatment. The mean body weight gain for males receiving AO-26 during treatment and the body weight gain for females before pairing for mating showed no adverse effect of treatment. During gestation females receiving 100 mg/kg/day showed slightly low weight gain; this was not evident at 25 and 50 mg/kg/day. Body weight gain during lactation was unaffected by treatment with AO-26. Food consumption for males and females for the two weeks before pairing and for females during gestation and lactation was unaffected by treatment.

There was no effect of treatment on estrous cycles, pre-coital interval, mating performance, fertility, gestation length and gestation length were unaffected by treatment. After 4 weeks of treatment males that received 50 or 100 mg/kg/day had high adjusted mean heart weight when compared with Controls (p<0.01); there was no dose response. Macroscopic examination of males after 4 weeks of treatment and females on Day 13 of lactation did not reveal any findings that could be attributed to treatment with AO-26. Myocardial degeneration associated with or without inflammatory cell infiltrate was seen in

the heart of both male and females that received 25, 50 or 100 mg/kg/day.

F1 responses

There were no clinical signs that could be attributed to parental treatment. The mean number of implantations at 100 mg/kg/day was slightly low when compared with Controls, however from Day 1 of age the live litter size was essentially similar across the groups. Offspring clinical condition, survival, body weight, sex ratio, ano-genital distance and macropathology were unaffected by parental treatment.

Conclusion

Treatment of AO-26 to parental animals at 25, 50 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility or macropathology. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment. Adverse findings were limited to an increase in heart weight for males that received 50 or 100 mg/kg/day and this correlated with histopathology which showed myocardial degeneration associated with or without inflammatory cell infiltrate which was seen in the heart of both male and females that received 25, 50 or 100 mg/kg/day. It was therefore concluded that the No-observed-adverse-effect-level (NOAEL) for reproductive performance was 100 mg/kg/day; however a NOAEL for parental toxicity was not established.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Justification for classification or non-classification

Treatment of AO-26 to parental animals at 25, 50 or 100 mg/kg/day for two weeks before pairing, during pairing and then up to termination of the males after 4 weeks of treatment and females on Day 13 of lactation was generally well tolerated. There was no adverse effect on parental clinical condition, body weight performance, food consumption, estrous cycles, mating performance, fertility or macropathology. The clinical condition, body weight and survival of the subsequent F1 offspring were also unaffected by parental treatment.

As no adverse effects were observed for reproductive performance the test substance AO-26 is not classified for reproductive toxicity.