4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2016 - 13 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

28 July 2015

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

EC Number:

299-434-3

EC Name:

4,4'-thiodiethylene hydrogen -2-octadecenylsuccinate

Cas Number:

93882-40-7

Molecular formula:

C48H86O8S

IUPAC Name:

2-octadecenylsuccinic anhydride, thiodiethanol esterification products

Test material form:

solid

Remarks:

Waxy Solid

Specific details on test material used for the study:

Test substance code in the study was EXP1504385

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Crl:CD(SD) Sprague Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test Animals
- Source: Charles River (UK) Margate, Kent UK
- Age at study initiation: males approximately 7-8 weeks old, females approximately 8-9 weeks old
- Weight at study initiation: males were between 247-311g, females were between 182-230g
- Fasting period before study: N/A
- Housing: Animals were initially housed 2 or 3 per cage by sex. A few days prior to mating the males were transferred to individual cages and after mating they were re-housed with their original cage mates. Once mated the females were individually housed.
- Diet (e.g. Ad libitum) SDS VRF-1 breeder diet (special diet services)
- Water (e.g. ad libitu): ad libitum
- Acclimation period: up to 2 weeks

Enviormental Conditions
- Temperature (°C): 18 - 21
- Humidity (%): 28-69
- Air Changes: minimum of 10 air changes per hour
- Photoperiod (hrs dark / hrs light): A 12 hour light/dark cycle

In-Life Dates: From: To: From: 21 Mar 2016 until 07 May 2016

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

-Doses: administered orally by gavage at a dose volume of 4 mL/kg body weight.
-Volume determined on each day by the weight of that animal as measured at the time of
administration, except during late gestation: from Day 16 until parturition was complete, the dose volume
was determined by the weight of the animal on Day 16 of gestation.

Details on mating procedure:

Pairings were on a one male to one female basis and animals were paired in numerical order within groups. A few days prior to initiation of mating, the males were separated into individual cages. On pairing, each female was transferred to the cage of its appropriate co-group male after 7 pm, where it remained until mating had occurred or 14 nights had elapsed.

Vaginal lavages were taken daily each morning from the day of pairing until mating had occurred and the stage of oestrus observed in each lavage was recorded. The day of detection of a copulatory plug in-situ and/or of sperm in the lavage was designated as Day 0 of gestation. The time taken for each female to show a positive mating sign was evaluated.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by ICP-OES using a validated analytical procedure (AP No. 435446).
Analyses performed from samples collected during week 1 and 4.

Duration of treatment / exposure:

Males: dosed once daily for 4 weeks, starting 2 weeks prior to mating.
Females: dosed once daily from 2 weeks prior to mating, throughout mating, gestation and parturition to
Day 4 of lactation.

Frequency of treatment:

Daily

Details on study schedule:

Study Initiation Date: 09 Mar 2016
Initiation of Dosing: 21 Mar 2016
Completion of In-life: 07 May 2016
Experimental Start Date: 14 Mar 2016
Experimental Completion Date: 13 Dec 2016

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

Males = 10 per dose group
Females = 10 per dose group

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: The oral route of administration was selected for this study as this route was
defined by the Sponsor as a possible route of human exposure.
The dose levels were selected based on information provided by the Sponsor from a 28 Day Repeated
Dose Oral Toxicity Study in the Sprague Dawley Rat with a 14 Day Recovery. In this study, the high
dose level of 1000 mg/kg/day was considered to be not appropriate based on clinical signs and effects
observed in the adrenals glands and stomach along with exudative inflammation of the nasal cavity.
The no observed adverse effect level (NOAEL) was considered to be 300 mg/kg/day based on the lack of
any remarkable toxicological effects other than alterations in several clinical pathology parameters. Due
to the increased dosing duration in study 497804 and the additional physiological stress expected in the females during pregnancy, parturition and lactation it was considered that 450 mg/kg/day was a suitable
high dose level as this was above the established NOAEL and was expected to produce some toxic
effects, but not excessive lethality that would prevent meaningful evaluation. The mid-dose level of
150 mg/kg/day was expected to produce minimal to moderate toxic effects and the low-dose level of
50 mg/kg/day to produce no observable indications of toxicity.

Examinations

Parental animals: Observations and examinations:

Observations and examinations performed and frequency
-Detailed Clinical Observations: Once each week, starting at week -1.
-Postdose Observations: Performed regularly throughout day. Animals were examined for reaction to treatment and the onset, intensity and duration of these signs.
-Body Weights: Recorded weekly during the pretreatment and daily during the dosing period, but reported weekly for the premating phase and also for Days 0, 7, 14 and 20 of gestation and Days 1 and 4 of lactation.
-Food Consumption: Recorded weekly for males and females until pairing for mating starting week -1. After this only mated female had food consumption recorded over Days 0 -7, 7 -14 and 14 -20 of gestation and Days 0 -4 of lactation.

Litter observations:

-Observations of Females with Litters during in Lactation: Day of birth of the litters was classified as Day 0 of lactation. The number of dead and live pups were recorded after completion of parturition. Live pups were examined daily for the presence of milk in the stomach and for any externally visible abnormalities. Each litter was weighed en masse (by sex) on Days 1 and 4 of lactation. Where practicable, any pups that were found dead or were killed during lactation were sexed and examined for the presence of milk in the stomach and for any externally visible abnormalities. Deficiencies in maternal care were recorded. The following were assessed: inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups, or apparently inadequate lactation or feeding.

Postmortem examinations (parental animals):

Gross Pathology, histopathology and specified organ weights

Postmortem examinations (offspring):

Pups were checked for the presence of any externally visible abnormalities. Externally
normal pups were discarded

Statistics:

Numerical data collected on scheduled occasions for the listed variables were constructed (as applicable) and analysed as indicated according to relevant classification variables (e.g., sex, occasion). Descriptive statistics: number, mean and standard deviation (or %CV and SE
when deemed appropriate) were reported wherever possible. Inferential statistics were performed as indicated when there were three or more non-missing values in each group, and were not performed on semi-quantitative data.

The following pairwise comparisons were made:
Group 1 vs. Group 2
Group 1 vs. Group 3
Group 1 vs. Group 4

Reproductive indices:

Fertility index (male and female) and Gestation index

Offspring viability indices:

birth Index, live birth index and viability index

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were a number of clinical observations recorded immediately postdose that were not present in the control group:

• Salivation: for 5 males and 6 females at 450 mg/kg/day, on a small number of occasions from Day 17 of treatment;
• Pedaling: for 3 males and 5 females at 450 mg/kg/day, on a small number of occasions from Day 24/25 of treatment;
• Ploughing: a dose-related increase in incidence was noted at 150 mg/kg/day (4 males and 6 females from Day 22) and 450 mg/kg/day (all animals from Day 20/22). A single female at 50 mg/kg/day was affected on Day 25 only.

Based on the findings being observed immediately after dosing, on a small number of occasions and generally not sustained throughout the treatment period these transient findings are considered not to be adverse.
The remaining clinical observations were considered not to be attributable to treatment with the Substance due to lack of a dose response, similarity in all groups or isolated incidences.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating Performance, Fertility, Duration of Gestation and Overall Litter
Performance
There were no treatment-related effects on any reproductive endpoint evaluated at dose levels up to 450 mg/kg/day including mating performance, fertility, pregnancy and litter performance.

The group mean time to mating was similar in all groups and only 1 pairing at 450 mg/kg/day showed evidence of mating that did not lead to a pregnancy. Fertility indices were at least 90%, with 1 pairing at each of 150 and 450 mg/kg/day not resulting in pregnancy. These findings are considered to be within normal variation.

The mean duration of gestation was similar in all groups and a gestation index of 100% was recorded for all groups.

Litter performance, in terms of group mean numbers of corpora lutea, implants, pups born and alive through to Day 4 of lactation. Observations were similar in dose groups when compared to the control groups.

Litter Survival and Litter Weights by Sex

Birth, live birth and pup viability indices were similar in all groups. While no control females lost more than 2 pups at birth (Birth Index) and 1 or 2 did in each group receiving the test Substance, was considered to be within normal variation and was not associated with a decrease in the group mean number of pups born.

Group mean litter weights and pup weights by sex were similar in all groups

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 450 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 450 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

limited by highest dose tested in parents

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Applicant's summary and conclusion

Conclusions:

In conclusion, administration of the Substance by once daily oral gavage was well tolerated in rats for approximately 4 or 7 weeks (males and females, respectively, including premating, mating, gestation and early lactation phases) up to 450 mg/kg/day. There were no effects on reproduction and development. The no-observed-effect level (NOEL) for reproduction and development was considered to be 450 mg/kg/day.

Following administration of the substance up to 450 mg/kg/day, there were no unscheduled deaths, effects on bodyweights or food consumption, and no test item-related gross findings, organ weight changes or microscopic findings observed. Minor clinical signs (salivation, ploughing and pedalling) were observed and are considered to be secondary to local irritation by the dosing solution rather than systemic toxicity. Based on these results, the no-observed-adverse-effect level (NOAEL) for systemic effects, in adult animals, was considered to be 450 mg/kg/day, which was the highest level tested in this study.

Executive summary:

The objective of this study was to provide initial information on possible effects on reproduction and/or development from repeated exposure of Crl:CD(SD) male and female rats to the registered Substance beginning before cohabitation, through mating and continuing for at least 28 days (male rats) or through parturition until at least Day 4 of lactation (female rats).

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight gains, food consumption, mating performance, duration of gestation, reproductive indices, litter data, gross necropsy findings, organ weights and histopathologic examinations.

Treatment with the Substance was associated with salivation and pedaling behaviour at 450 mg/kg/day on a small number of occasions in 5-6 animals/sex and 3-5 animals/sex, respectively. Additionally ploughing was observed, on a small number of occasions, in all the animals at 450 mg/kg/day, 4-6 animals per sex at 150 mg/kg/day and in one female in one occasion at 50mg/kg/day. These effects were observed from study Day 17 -24 and immediately postdose. These transient findings are considered not to be adverse.

There were no effects of treatment on any other end points.

In conclusion, administration of the Substance by once daily oral gavage was well tolerated in rats for approximately 4 or 7 weeks (males and females, respectively, including premating, mating, gestation and early lactation phases) up to 450 mg/kg/day. There were no effects on reproduction and development. The no-observed-effect level (NOEL) for reproduction and development was considered to be 450 mg/kg/day.

Following administration of the Substance up to 450 mg/kg/day, there were no unscheduled deaths, effects on bodyweights or food consumption, and no test item-related gross findings, organ weight changes or microscopic findings observed. Minor clinical signs (salivation, ploughing and pedalling) were observed and are considered to be secondary tolocal irritation by the dosing solution rather than systemic toxicity. Based on these results, the no-observed-adverse-effect level (NOAEL) for systemic effects, in adult animals, was considered to be 450 mg/kg/day, which was the highest level tested in this study