2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 July to 06 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD guideline compliant study with no deviations

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 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Di-TMP

IUPAC Name:

Di-TMP

Constituent 2

Reference substance name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

EC Number:

245-509-0

EC Name:

2,2'-[oxybis(methylene)]bis[2-ethylpropane-1,3-diol]

Cas Number:

23235-61-2

Molecular formula:

C12H26O5

IUPAC Name:

2-ethyl-2-{[2-ethyl-3-hydroxy-2-(hydroxymethyl)propoxy]methyl}propane-1,3-diol

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): Di-TMP
- Substance type: flakes
- Analytical purity: 98.9%
- Lot/batch No.: 4474131
- Expiration date of the lot/batch: 06 October 2016
- Storage condition of test material: ambient, in the dark

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Margate, UK
- Age at study initiation: 9 weeks old
- Weight at study initiation: 177-269 g on arrival; 197-307 g at the initiation of dosing
- Housing: 2 per cage in appropriately sized suspended polycarbonate/polypropylene cages with stainless steel grid tops and solid bottoms. Bedding material was sterilised white wood shavings, and enrichment (devices for hiding in and an object for chewing)
- Diet (e.g. ad libitum): SDS VRF-1 breeder diet, ad libitum
- Water (e.g. ad libitum): water from the public supply, ad libitum
- Acclimation period: 3-5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-21°C
- Humidity (%): 42-62%
- Air changes (per hr): ten or more with 100% fresh air
- Photoperiod (hrs dark / hrs light): 12 hour cycle

IN-LIFE DATES: From: 27 July 2015 To: 12 August 2015

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose volume was 4 mL/kg, and the concentration of the test substance in the vehicle was 0, 25, 75 and 250 mg/mL. Dose volumes were based on the most recent body weight recorded for each animal. The control item, corn oil, was aliquoted weekly, stored in a refrigerator set to maintain 4°C and dispensed daily. The prepared control item was removed from the refrigerator and stirred for at least 30 minutes prior to dosing, and continuously during dosing. Prior to formulation, the test item was ground in a mortar, since it was supplied as flakes. The required amount of ground test item was weighed into a pre-labelled container according to instructions from the formulation computerised system (Dispense 8). The appropriate amount of vehicle was then added to the container and the formulation was magnetically stirred until visibly homogenous. The dosing formulations were prepared weekly, stored in a refrigerator set to maintain 4°C, and dispensed daily. The dosing formulations were removed from the refrigerator and were stirred for at least 30 minutes before dosing and continuously during dosing.

Stability analyses performed previously in conjunction with Charles River Study No. 434348 demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study for at least 8 days at 2-8°C, in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed by Gas Chromatography using a validated analytical procedure. Duplicate top, middle and bottom samples (middle only for control) of 0.1 mL for each sampling time point were collected and sent to the analytical laboratory for analysis. Triplicate sets of top, middle and bottom samples (middle only for control) of the same volume were also collected and retained as backup samples. Concentration results were considered acceptable if mean sample concentration results were within 10% of theoretical concentration and individual sample concentrations were within 15% of theoretical concentration. Homogeneity results were considered acceptable if the relative standard deviation of the mean value at each sampling location was ≤10%.

Details on mating procedure:

Not applicable - time mated females were obtained direct from the supplier.

Duration of treatment / exposure:

From Days 6-19 of gestation

Frequency of treatment:

Once daily

Duration of test:

Day 0 (mating) to Day 20 of gestation (scheduled termination)

No. of animals per sex per dose:

24 females per dose

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen by the Sponsor following review of data from a 28 day repeated dose study of DiTMP in rats. On that study, tubular basophilia and inflammatory cell infiltration in the kidney were noted in males at all dose levels (40, 200 or 1000 mg/kg/day); however, these findings did not follow a clear dose-related pattern, were of little toxicological significance and were not noted in females. The NOAEL was therefore considered to be 1000 mg/kg bw/d.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
All animals were observed twice daily, once in the morning and once towards the end of the working day throughout the study for general health/mortality and moribundity. Animals were observed prior to dosing and regularly throughout the day for reaction to treatment on each day of dosing. The onset, intensity and duration of these any signs were recorded, with particular attention paid to the first hour after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
Animals were given a detailed examination once pretreatment and daily from Day 6 of gestation.

BODY WEIGHT: Yes
Individual body weights were recorded on Day 4 of gestation and daily from Days 6 to 20 of gestation.

FOOD CONSUMPTION: Yes
Food consumption was quantitatively measured daily throughout the study, beginning on Day 4 of gestation (first measured quantity given on Day 3 of gestation).

WATER CONSUMPTION: Yes
Water consumption was monitored by visual inspection of the water bottles only. As no intergroup differences were noted, consumption was not measured by weight.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: All adult animals were given an external examination, and then the contents of the cranial, thoracic and abdominal cavities were examined macroscopically, with representative samples of abnormal tissues being fixed in neutral buffered 10% formalin. The reproductive tract was dissected out and examined

Ovaries and uterine content:

The reproductive tract was dissected from the abdominal cavity. The gravid uterus weight was recorded. The ovaries and uterus were examined for number and distribution of: Corpora lutea; Implantation sites; Placentae (size, colour or shape) – only abnormalities were recorded; Live and Dead Fetuses; Early and Late Resorptions

Fetal examinations:

External abnormalities - Each fetus was examined for external abnormalities. Late resorptions and dead fetuses were examined for external abnormalities to the extent possible. Each implant was classified as being live, or a dead fetus (dead full term fetus that showed no signs of maceration), or a late embryonic death (macerated tissue identifiable as an embryofetus, with recognizable external features such as tail, limbs, mouth and nares present; attached to distinct identifiable placentae), or an early embryonic death (discrete, formless, discoloured tissue mass attached to the internal uterine wall; may have been of varying size).

Visceral examinations and sex - Half of the viable foetuses from each uterus were fixed in methylated ethyl alcohol, examined internally for sex and eviscerated following fixation, the viscera were not examined from fetuses prior to disposal. The remaining half of viable fetuses from each uterus were fixed in Bouins’ fluid. The foetuses fixed in Bouins’ fluid were examined for soft tissue abnormalities and sex by a freehand sectioning technique derived from Wilson.

Skeletal examination - The eviscerated carcasses were macerated in potassium hydroxide, the skeletons stained with Alizarin Red S, then the fetuses cleared with aqueous glycerol solutions. These preparations were examined for the presence of skeletal abnormalities and for the extent of ossification.

Statistics:

Means and standard deviations were calculated for body weight, food consumption and selected pregnancy data. Numerical data collected on scheduled occasions for the listed variables were analysed as indicated according to relevant classification variables (e.g., sex, occasion). Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) are reported whenever possible. Inferential statistics were performed for body weight, food consumption and body weight change when possible, but excluded semi-quantitative data and any group with less than 3 observations.

ANOVA - Levene’s test was used to assess the homogeneity of group variance parametric assumption at the 5% significance level. Datasets with at least three groups was compared using an overall one-way ANOVA F-test or Kruskal-Wallis test (if parametric assumptions were not met) at the 5% significance level. The above pairwise comparisons were conducted using a two-sided Dunnett’s or Dunn’s test, respectively, if the overall test is significant. Datasets with two groups were compared using a two-sided t-test or Wilcoxon Rank-Sum test, respectively. All significant pairwise comparisons were reported at the 0.1, 1 and 5% significance levels.

Indices:

Not applicable

Historical control data:

Available at the test facility

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no mortalities during the study. An increased incidence of ploughing behaviour (animal burrowing through bedding with its head) was noted in all treated groups; this observation was absent in Controls. This finding was transient, being noted immediately post dosing, and was no longer evident 1 hour after dosing, and was generally noted on 2-3 occasions between Days 14-20 of gestation in affected animals. All other clinical observations were considered to be incidental background findings commonly observed in this species and unrelated to treatment with DiTMP.
There were no effects on group mean body weights, group mean food consumption, and there were no abnormal findings detected at necropsy.

Pregnancy performance parameters and fetal weights were similar between all groups. Slight intergroup differences in embryonic deaths and fetal weights were noted between groups; however, these were considered to be incidental as they were within normal variation and were too small to be attributed to treatment with DiTMP.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes. Remark: In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls

Details on embryotoxic / teratogenic effects:
In all treated groups, dose-related increased incidences of misshapen scapula(e) were noted when compared with Controls (14 fetuses in 10 litters at 100 mg/kg/day, 17 fetuses in 9 litters at 300 mg/kg/day and 23 fetuses in 10 litters at 1000 mg/kg/day, compared with 6 fetuses in 4 Control litters).
At 1000 mg/kg/day, increased incidences of unossified hyoid bone and 5th metacarpal(s) were noted when compared to Controls. Decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches were also observed in this group, when compared with Controls.
Renal pelvic dilation, which was noted in a small number of fetuses and litters at 1000 mg/kg/day, was considered unlikely to be related to treatment due to the low incidence and since the number of fetuses and litters affected is within background control ranges at the Test Facility.
The type and distribution of major fetal abnormalities and all other minor fetal abnormalities and variants and skeletal ossification parameters did not indicate any association with treatment. Slight intergroup differences were considered to be incidental and unrelated to treatment with DiTMP.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

< 100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

skeletal malformations

2

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

skeletal malformations

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

Dose formulation analyses: Analysed Group 2, 3 and 4 formulations from Study Weeks 1 and 2 were found to be within the concentration acceptance criteria of ±10% of theoretical concentration (actual mean concentration range -7% to +1.7% of theoretical concentration). A low relative standard deviations of ≤4.5% also indicated that all formulations were homogenous. The absence of DiTMP from analysed control formulations was also confirmed.

Summary of Group Incidences of Treatment Related Clinical Observations

	Dose Group/Dose Level (mg/kg bw/d)
Observation	1 (0)	2 (100)	3 (300)	4 (1000)
Ploughing behaviour	0/24	11/24	14/24	24/24

Group Incidences of Minor Fetal Abnormalities and Variants

Abnormality/Variant	Group/Dose Level (mg/kg bw/d)
	1 (0)	2 (100)	3 (300)	4 (1000)
	Incidence of Fetuses (Litters)
Skeletal
Basisphenoid incompletely ossified, bilateral jugal misshapen	0	0	1(1)	0
Basioccipital misshapen	0	0	0	1(1)
Cranial bone(s) discrete unossified/incompletely ossified area(s)	3(2)	0	1(1)	2(1)
Cervical vertebral arch(es) increased ossification	11(7)	5(4)	12(8)	8(7)
Scapula(e) misshapen	6(4)	14(10)	17(9)	23(10)
Bilateral humerus incompletely ossified	0	0	0	1(1)
Sternebra bifurcated, xiphoid cartilage incomplete	0	1(1)	0	0
Rib(s) minimally kinked	0	1(1)	1(1)	2(2)
Rib(s) incompletely ossified	0	0	0	1(1)
Additional ossified area arising from sternebra	2(2)	0	1(1)	1(1)
Rib(s) costal cartilages asymmetrically aligned	7(7)	7(6)	6(6)	1(1)
Rib(s) costal cartilage(s) not attached to sternum	4(3)	2(1)	2(2)	1(1)
Pelvic girdle cranial displacement: Unilateral	1(1)	0	0	0
Pelvic girdle caudal displacement
Unilateral:	0	0	1(1)	0
Bilateral:	0	1(1)	0	0
Number with minor abnormality/variant	31(18)	29(15)	37(19)	36(16)
Total number examined skeletally	164(24)	150(24)	157(24)	161(24)
Number of ribs
13th vestigial rib(s)	4(3)	2(2)	0	1(1)
13th reduced rib(s)	2(2)	0	1(1)	3(1)
13 complete rib(s)	149(24)	142(24)	152(24)	155(24)
Vestigial supernumerary rib(s) on 1st lumbar vertebra	9(6)	6(5)	3(3)	2(2)
Reduced supernumerary rib(s) on 1st lumbar vertebra	0	0	1(1)	0

Group Incidences of Skeletal Ossification Parameters

Parameter	Group/Dose Level (mg/kg bw/d)
	1 (0)	2 (100)	3 (300)	4 (1000)
	Incidence of Fetuses (Litters)
Incomplete ossification affecting:
≥4 skull bones	6(4)	2(2)	5(3)	7(5)
≤3 skull bones	26(14)	14(7)	16(9)	21(12)
Cervical vertebral arch(es)	1(1)	1(1)	1(1)	0
Thoracic vertebral centrum(a)	9(7)	12(9)	8(6)	8(7)
Lumbar vertebral arch(es)	0	0	1(1)	0
Lumbar vertebral centrum(a)	0	0	1(1)	0
Pubis/es	5(3)	5(4)	1(1)	2(2)
Ischium/a	1(1)	0	3(2)	0
Sacral vertebral arch(es)	18(3)	6(5)	6(3)	8(6)
2nd and/or 4th metacarpal(s)	5(4)	1(1)	3(3)	1(1)
Unossified:
Hyoid	13(9)	4(4)	8(4)	20(12)
5th metacarpal(s)	29(13)	20(10)	29(14)	41(13)
5th metatarsal(s)	0	1(1)	1(1)	0
Ossified:
Anterior arch of atlas	51(17)	51(20)	41(13)	36(17)
>2 cervical vertebral centra	15(11)	13(8)	17(8)	10(8)
One or more sacrocaudal vertebrae with connection between centrum and arch(es)	46(19)	46(21)	53(17)	35(16)
Phalangeal elements	23(9)	33(12)	26(11)	13(8)
Mean number of caudal vertebral centra	4.1	4.0	4.1	3.8
Number of sternebrae retarded:
0	69(24)	71(22)	85(21)	60(19)
1	75(22)	58(21)	59(21)	70(22)
2	18(11)	12(11)	11(9)	27(14)
>2	2(2)	9(4)	2(2)	4(4)
Total number examined skeletally	164(24)	150(24)	157(24)	161(24)

Applicant's summary and conclusion

Conclusions:

The NOAEL for maternal toxicity was considered to be 1000 mg/kg bw/d, however a developmental NOAEL could not be established.

Executive summary:

The potential for DiTMP to cause prenatal developmental toxicity was investigated in pregnant Sprague-Dawley rats, according to OECD 414. DiTMP was administered to groups of 24 time-mated rats by gavage at doses of 0, 100, 300 or 1000 mg/kg bw/d in corn oil. Animals were dosed over Days 6-19, inclusive, of gestation (where the day of detection of mating was Day 0 of gestation) and regularly checked for clinical signs of toxicity, body weight and food consumption performance and were killed on Day 20 of gestation for examination of pregnancies and embryofetal development.

Dosing with DiTMP was not associated with any maternal body weight or food consumption effects or gross necropsy findings. A dose-related increased incidence of ploughing behaviour was noted in all treated groups when compared to controls; however, as this findings was transient, noted only on a few occasions in each individual and is thought to be a reaction to unpleasant taste of dosing formulations, it was considered not to be adverse at any level.

Pregnancy performance and fetal weights in all groups were similar, and there were no major fetal abnormalities which were considered to be related to treatment with DiTMP. In all treated groups, a dose-related increased incidence of misshapen scapula(e) was noted when compared with controls. This finding is difficult to interpret as it had only recently been noted at the Test Facility in a small number of studies at low incidence, therefore background control data on this finding is limited, and the incidences in all treated groups on this study are above the background range. The significance of this finding could therefore not be determined. At 1000 mg/kg/day, a general trend toward delayed ossification was noted (which included increased incidences of unossified hyoid bones and 5th metacarpals and decreased incidences of ossified anterior atlas, cervical vertebral centra, phalangeal elements and sacrocaudal vertebrae with connections between centrum and arches) when compared with Controls. It was therefore concluded from the results of this study, that the maternal NOAEL was 1000 mg/kg bw/d. A developmental NOAEL cannot be established due to an increased and dose-related incidence of misshapen scapulae at all dose levels and a trend towards delayed ossification at 1000 mg/kg bw/d.