Pentaerythritol tetrakis(3-mercaptopropionate)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-06-19 - 2015-01-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study, GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 January 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Pentaerythritol tetrakis(3-mercaptopropionate)

EC Number:

231-472-8

EC Name:

Pentaerythritol tetrakis(3-mercaptopropionate)

Cas Number:

7575-23-7

Molecular formula:

C17H28O8S4

IUPAC Name:

3-[(3-sulfanylpropanoyl)oxy]-2,2-bis({[(3-sulfanylpropanoyl)oxy]methyl})propyl 3-sulfanylpropanoate

Details on test material:

- Name of test material (as cited in study report): PETMP
- Physical state: liquid
- Analytical purity: 97.4% w/w

Test animals

Species:

rat

Strain:

other: Crl:CD(SD) IGS BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: young adult
- Weight at study initiation: 190 to 264 g (on arrival)
- Fasting period before study: no
- Housing: ndividually in solid-floor polypropylene cages with stainless steel mesh lids furnished with softwood flakes (Datesand Ltd., Cheshire, UK), containign environmental enrichment
- Diet (e.g. ad libitum): pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan UK, Oxon, UK), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 1 - 2 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Animals were delivered in two batches containing time mated females prior to Day 3 (Day 0 or Day 1) of gestation.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
emulsion in Corn Oil

VEHICLE
- Concentration in vehicle: 12.5, 30, 75/50 mg a.i./L
- Amount of vehicle (if gavage): dose volume 4 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentrations were determined by HPLC with UV detection (215 nm) using an external standard.
Samples were extracted with acetonitrile (ultra-sonicated for 15 min and then centrifuged at 4500 rpm for 10 min).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Day 3 to Day 19 of gestation

Frequency of treatment:

daily

Duration of test:

animals were killed on Day 20 of gestation

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on dose range finding study

Examinations

Maternal examinations:

Morbidity/Mortality Inspection
Twice daily, early and late during the working period.

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily during the gestation period; immediately before and soon after dosing, half an hour post dosing, one hour post dosing and approximately four hours post dosing

BODY WEIGHT: Yes
- Time schedule for examinations: Day 3 (before start of treatment), and on Days 4, 5, 8, 11, 14 and 17 of gestation. Body weights were also recorded for surviving animals at terminal kill (Day 20)

FOOD CONSUMPTION: Yes
-Day 3, 5, 8, 11, 14, 17 and 20 of gestation

WATER CONSUMPTION: Yes
Monitored daily by visual inspection of water bottles

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Full external and internal macroscopic examination of adult females. Any macroscopic abnormalities were recorded.
As macroscopic changes were noted in the stomach of the decedent females; the stomach from all females surviving to termination were retained at necropsy. The ovaries and uteri of pregnant females were removed, examined and the following data recorded.

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- fetal weight
- placental weight
- fetal sex

Statistics:

Group mean values were calculated to include data from all females with live fetuses on Day 20 of gestation.
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.

Indices:

Pre and Post Implantation Loss
Sex Ratio

Historical control data:

Historical control data for Sprague-Dawley Crl:CD® (SD) IGS BR Rat are provided in the study report for the following parameters: Gestation Body Weights, Gestation Food Consumption, Litter Data, External Fetal Observations, Visceral Fetal Findings, Skeletal Fetal Findings

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality
One female treated with 300/200 mg a.i./kg bw/day was killed in extremis on Day 8 due to the persistence of clinical observations of clonic convulsions, splayed gait, occasional body tremors and ataxia. Multiple raised areas were apparent in the non-glandular region of the stomach at necropsy.
One female treated with 120 mg a.i./kg bw/day was found dead on Day 13 following observations of clonic convulsions, decreased respiratory rate, laboured respiration, gasping respiration, staining of the fur and increased salivation immediately post dose. This animal was found to have an oily red fluid in the thoracic cavity at necropsy, and sloughing of the glandular region of the stomach was also apparent. The death of this animal was considered to have been caused by a dosing trauma or aspiration of the test item when dosed.

Clinical Observations
A number of females treated with 300/200 mg a.i./kg bw/day had transient episodes of occasional body tremors, tonic and clonic convulsions, hunched posture, ataxia, lethargy, piloerection, splayed gait and, laboured respiration and decreased respiratory rate up to Day 11. Thereafter no such observations were apparent.
The majority of animals at this dose level also had episodes of increased salivation post-dose from Day 8 onwards. Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance. No such effects were detected in females surviving to necropsy at 50 or 120 mg a.i./kg bw/day.

Body Weight
Females treated with 300/200 mg a.i./kg bw/day showed group mean body weight losses between Day 3 and Day 4 with differences from control attaining statistical significance. Recovery was evident thereafter although group mean body weight gains were slightly lower than control from Day 14 onwards. Consequently, cumulative body weight gains to Day 5 were statistically significantly lower than controls and thereafter remained lower than control throughout the treatment period.
Gravid uterus weight for females treated with 300/200 mg a.i./kg bw/day was slightly lower than controls however statistical significance was not achieved. Body weight gain when adjusted for the contribution of the gravid uterus was also slightly lower than controls. No such effects were detected in females treated with at 50 or 120 mg a.i./kg bw/day

Food Consumption
Food consumption for females treated with 300/200 mg a.i./kg bw/day was statistically significantly lower than controls to Day 8 of the study and generally remained lower than controls throughout the remainder of the treatment period. No such effects were detected in females treated with 50 or 120 mg a.i./kg bw/day.

Water Consumption
Daily visual inspection of water bottles did not reveal any overt intergroup differences.

Post Mortem Studies
No macroscopic abnormalities were detected in any female surviving to termination.

Effect levels (maternal animals)

1

Dose descriptor:

NOEL

Effect level:

120 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

2

Dose descriptor:

NOEL

Effect level:

120 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Fetal Weights
The mean male fetal weight for fetuses from females treated with 300/200 mg a.i./kg bw/day was statistically significantly lower than controls. The mean female fetal weight and mean fetal weight for both sexes were also slightly lower than controls, however, statistical significance was not achieved.
There were no obvious adverse effects of maternal treatment on litter data as assessed by the number of implantations, early and late embryonic/fetal deaths and live fetuses or sex ratio, as assessed by percentage male.

Fetal Examination
Increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae achieved statistical significance in comparison to controls for females treated with 300/200 mg a.i./kg bw/day. Increased incidence of incomplete ossification of the interparietal and occiptal (supra-occiptal), dumb-bell shaped thoracic centrum and unossified metacarpals were also apparent when compared to controls although the magnitude of these differences did not achieve statistical significance.
A lower incidence of the number of fetuses showing ossification of the 1st metatarsal attained statistical significance for females treated with 300/200 or 120 mg a.i./kg bw/day Visceral examination of the fetuses from females treated with 200 mg a.i./kg bw/day also revealed increased incidence of absent renal papilla with differences from control attaining statistical significance. Increased incidence of increased cavitation of the renal pelvis was also apparent, however, statistical significance was not achieved.

Effect levels (fetuses)

Remarks on result:

not measured/tested

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Stability of the test item formulations

Measured concentrations of the test item formulations were within 100-107% of nominal. Formulations were stable when kept for 12 d at 4°C.

Group Mean Body Weight Values

Dose Level (mg a.i./kg bw/day)		Body Weight (g) at Day of Gestation
		3	4	5	8	11	14	17	20
0 (Control)	mean	244.8	249.4	255.0	270.2	289.9	309.1	342.8	386.2
	sd	22.9	23.5	24.1	24.5	27.2	28.9	33.7	39.2
	n	24	24	24	24	24	24	24	24
50	mean	242.4	247.1	253.2	268.4	286.5	305.2	338.4	382.5
	sd	12.4	12.1	12.3	13.5	16.2	16.0	18.2	17.8
	n	24	24	24	24	24	24	24	24
120	mean	243.3	247.0	253.5	268.5	289.9	306.3	337.8	381.4
	sd	12.6	12.5	13.0	15.3	16.5	20.5	23.4	27.4
	n	23	23	23	23	23	23	23	23
300/200	mean	242.5	242.2	248.0	263.5	283.7	300.7	330.3	370.3
	sd	18.2	21.2	22.7	22.4	22.2	24.7	28.5	34.2
	n	23	23	23	23	23	23	23	23

 

Group Mean Body Weight Change

Dose Level (mg a.i./kg bw/day)		Cumulative Body Weight Change (g) from Day 3 of Gestation
		4	5	8	11	14	17	20
0 (Control)	mean	4.7	10.3	25.4	45.2	64.4	98.0	141.5
	sd	3.6	3.9	5.5	8.0	9.6	14.6	21.8
	n	24	24	24	24	24	24	24
50	mean	4.7	10.8	26.0	44.0	62.8	96.0	140.1
	sd	2.5	3.3	4.1	8.2	8.3	11.0	12.4
	n	24	24	24	24	24	24	24
120	mean	3.7	10.1	25.1	46.5	63.0	94.5	138.0
	sd	2.7	2.7	5.0	7.1	11.9	15.2	21.1
	n	23	23	23	23	23	23	23
300/200	mean	-0.3**	5.5*	21.0	41.2	58.1	87.8	127.8
	sd	6.2	7.5	7.1	8.1	10.2	14.9	24.6
	n	23	23	23	23	23	23	23

 

Group Mean Gravid Uterus Weight and Adjusted Body Weight and Body Weight Change Values

Dose Level (mg a.i./kg bw/day)		Body Weight (g) on Days of Gestation		Body Weight Change (g) during Days of Gestation 3 – 20	Gravid Uterus Weight (g)	Adjusted to Body Weight (g) Day 20	Adjusted to Body Weight (g) Change 3 - 20
		3	20
0 (Control)	mean	244.8	386.2	141.5	83.115	303.1	58.3
	sd	22.9	39.2	21.8	15.272	26.6	10.5
	n	24	24	24	24	24	24
50	mean	242.4	382.5	140.1	82.959	299.6	57.2
	sd	12.4	17.8	12.4	7.565	18.1	12.9
	n	24	24	24	24	24	24
120	mean	243.3	381.4	138.0	81.329	300.1	56.8
	sd	12.6	27.4	21.1	11.972	21.6	15.0
	n	23	23	23	23	23	23
300/200	mean	242.5	370.3	127.8	74.138	296.2	53.7
	sd	18.2	34.2	24.6	23.279	26.5	12.8
	n	23	23	23	23	23	23

 

* Significantly different from control group p<0.05

** Significantly different from control group p<0.01

Applicant's summary and conclusion

Conclusions:

The oral administration of PETMP to pregnant rats by oral gavage during gestation at dose levels of 50, 120 and 300/200 mg/kg bw/day (incorporating a correction factor for 97.4 % purity), resulted in treatment-related effects for females treated with 300/200 mg a.i./kg bw/day with an associated effect on fetal growth.
The No Observed Effect Level (NOEL) for the pregnant females and the survival, growth and embryofetal development of the offspring was considered to be 120 mg a.i./kg bw/day.

Executive summary:

In a developmental toxicity study according to OECD Guideline 414 (22 January 2001) and EU method B.31 (30 May 2008) PETMP (97.4% a.i.) was administered was administered to 24 time mated femaleSprague-Dawley Crl:CD® (SD) IGS BR strain rats/dose in corn oilby gavageat dose levels of 0,50, 120, and 300/200 mg a.i./kg bw/day (incorporating a correction factor for 97.4% purity)from days 3 through 20 of gestation.

Adverse clinical signs were apparent in two animals treated with 300 mg a.i./kg bw/day following one dose therefore the dose level was reduced to 200 mg a.i./kg bw/day. Therefore, the first twelve animals of the high dose received the test item at 300 mg a.i./kg bw/day for one or two days prior to the reduction of the dose level; the remaining twelve animals were treated with 200 mg a.i./kg bw/day throughout dosing. Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

One female treated with 300/200 mg a.i./kg bw/day was killedin extremison Day 8 due to the persistence and severity of clinical observations. One female treated with 120 mg a.i./kg bw/day was found dead on Day 13. The death of this animal was considered to have been caused by a dosing trauma or aspiration of the test item when dosed.

Clinical observations of occasional body tremors, clonic convulsions, hunched posture, ataxia, lethargy, pilo-erection, splayed gait and, labored respiration and decreased respiratory rate were apparent transiently up to Day 11 in females treated with 300/200 mg a.i./kg bw/day.  No such effects were detected in females surviving to necropsy at 50 or 120 mg a.i./kg bw/day.

At 300/200 mg a.i./kg bw/day group mean body weight losses were apparent on Day 4 with an associated reduction in cumulative body weight gain being seen to Day 5 of treatment. Initial recovery of body weight gain was apparent thereafter, however, body weight performance from Day 14 onwards appeared to be lower than controls. Gravid uterus weight and body weight gain, when adjusted for the contribution of the gravid uterus were also slightly lower than controls. No such effects were detected in females treated with 50 or 120 mg a.i./kg bw/day.

Food consumption for females treated with 300/200 mg a.i./kg bw/day was generally lower than controls throughout the treatment period with differences from control attaining statistical significance to Day 8. No such effects were detected in females treated with 50 or 120 mg a.i./kg bw/day.

Daily visual inspection of water bottles did not reveal any overt intergroup differences.

No macroscopic abnormalities were detected in any female surviving to termination.

 

The maternal LOAEL is 200 mg a.i./kg bw/day, based on clinical signs, reduced body weight, reduced body weight gain, reduced food consumption and slightly lower gravid uterus weight (though not statistically significant). The maternal NOEL is 120 mg a.i./kg bw/day. 

 

The mean male fetal weight for fetuses from females treated with 200 mg a.i./kg bw/day was statistically significantly lower than controls. The mean female fetal weight and mean fetal weight for both sexes were also slightly lower than controls, however, statistical significance was not achieved.

Increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae achieved statistical significance in comparison to controls for females treated with 300/200 mg/kg bw/day. Increased incidence of incomplete ossification of the nasal, frontal, interparietal and occiptal (supra-occiptal), dumb-bell shaped thoracic centrum and unossified metacarpals were also apparent when compared to controls although the magnitude of these differences did not achieve statistical significance.

A lower incidence of the number of fetuses showing ossification of the 1st metatarsal attained statistical significance for females treated with 200 or 120 mg/kg bw/day A.I. In isolation and in the absence of any differences in a number of variants or a syndrome of variance, the intergroup difference at 120 mg a.i./kg bw/day was considered not to be toxicologically significant. Visceral examination of the fetuses from females treated with 200 mg a.i./kg bw/day also revealed increased incidence of absent renal papilla with differences from control attaining statistical significance. Increased incidence of increased cavitation of the renal pelvis was also apparent, however, statistical significance was not achieved.

 

The developmental LOAEL is 200 mg a.i./kg bw/day, based on lower mean male fetal weight, increased incidence of fetuses showing incomplete ossification of the thoracic centrum or less than four ossified caudal vertebrae, increased incidence of absent renal papilla.  The developmental NOEL is 120 mg/kg bw/day.

 

Based on Classification and Labelling guidance it can be postulated that classification as a developmental toxicant at either Category 1B or 2 may not be warranted because of the effects seen in adults with consequential effects on offspring development.

 

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study (OECD 414) in rat.