Phosphorous acid, mixed 2,4-bis(1,1-dimethylpropyl)phenyl and 4-(1,1-dimethylpropyl)phenyl triesters

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 October 2013 to 05 December 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed in accordance with OECD, EU & US EPA test guidelines in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline" below.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline" below.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

See "Principles of method if other than guideline" below.

Principles of method if other than guideline:

List of protocol deviations
1. On 16-November-2013 animals were observed but the data was not saved online. One control female (no. 17) had a late resorption (fetus no. 13) where no degree of autolysis,
crown-rump length measurement or external findings were recorded.
Evaluation: Sufficient data were available for a thorough evaluation.
2. From 27 November 2013 onwards, Lignocel S 8-15 (Rettenmaier & Sohne GmbH, Rosenberg, Germany) was used for sterilized sawdust bedding instead of the bedding specified in the protocol.
Evaluation: Both bedding types are suitable for use in this type of study.
3. Only the left ovary was preserved for animal no. 28 (1500 ppm).
Evaluation: Sufficient data were available for a thorough evaluation.
4. Organ weights were collected for all animals and not just from the pregnant animals.
Evaluation: This just represents additional information and does not negatively impact the
study.
The study integrity was not adversely affected by the deviations.

GLP compliance:

yes

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

EC Number:

700-485-5

Cas Number:

939402-02-5

Molecular formula:

Mixture of 4 components, the molecular formulae of which are: C33 H45 O3 P, C38 H55 O3 P, C43 H65 O3 P and C48 H75 O3 P

IUPAC Name:

Reaction mass of bis[2,4-bis(2-methylbutan-2-yl)phenyl] 4-(2-methylbutan-2-yl)phenyl phosphite and 2,4-bis(2-methylbutan-2-yl)phenyl bis[4-(2-methylbutan-2-yl)phenyl] phosphite and tris[4-(2-methylbutan-2-yl)phenyl] phosphite.

Test material form:

liquid: viscous

Details on test material:

Identification: DVS 005 (Weston 705T)
CAS Number: 939402-02-5
Description: Clear colourless viscous liquid (determined at WIL Research Europe B.V.)
Batch: MW3G18T701
Purity/Composition: 100%
Test substance storage: In refrigerator (2-8°C) under nitrogen, desiccated
Stable under storage conditions until: 30 November 2014 (expiry date)
Purity/composition correction factor required: No
Hygroscopic: Yes, desiccated
Volatile: No
Reactivity: Reactive to moisture
Test substance handling: Flush container with nitrogen after handling
Specific Gravity / Density: 1.0180 (25/15.5°C)
pH: 6.0
Stability at higher temperatures: Yes, maximum temperature: 70°C
Stability in diet: Stability in diet over 29 days at room temperature (range of 1500-15000 mg test substance/kg diet) was confirmed in Project 503366.

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

Test System Rat: Crl:WI(Han) (outbred, SPF-Quality).
Females were nulliparous, non-pregnant and untreated at initiation of the study. Stock male rats Crl:WI(Han) (outbred, SPF-Quality) were used for mating with the females. After mating these males were placed back in their stock and might be used in further studies.

Rationale: This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of developmental toxicants.

Source: Charles River Deutschland, Sulzfeld, Germany.
Number of animals: F0-generation: 88 females. F1-generation: 893 fetuses.
Age at delivery: Females were approximately 11 weeks
Acclimatization: At least 5 days prior to start of treatment.
Health inspection: Upon receipt of the animals.

Randomization: Upon detection of mating (Day 0 post-coitum), the females were distributed in a random sequence over the test groups. Females which were mated on the same day were classified in the same subgroup.
Identification: By indelible ink.

Conditions: Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle (lights on at 0700).
Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Pre-mating: During acclimatization, females were housed in groups of maximum 5 animals/cage in Macrolon plastic cages (MIV type, height 18 cm).
Mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm).
Post-coitum: Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
General: Sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France, from 06-26 November and Lignocel S 8-15, Rettenmaier & Sohne GmbH, Rosenberg, Germany from 27 November onwards) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied.
Diet: Free access to prepared diets. During acclimatization and pre-treatment (Day 0 to 6 post-coitum), animals had free access to the same diet (without the test substance) received from the supplier in pelleted form (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water: Free access to tap-water.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

Diet: Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Method: When necessary, the test substance was heated up to 70°C for a maximum of 6 hours before mixing with powder feed. The test substance was mixed without the use of a vehicle, directly with some powder feed (premix) and subsequently mixed with the bulk of the diet. Water (approximately 15% in total) was added to aid pelleting. The pellets were dried for approximately 24 hours at 35°C before storage. The control animals received similarly prepared pellets but without the test substance.

Frequency of preparation: Diets were prepared at least once every 15 days. The same diets were used for the two-generation study (Project 503367).
No correction was made for the purity/composition of the test substance.

Stability of prepared diets: Stability of the diets at 1500 and 15000 ppm for at least 29 days at room temperature and for at least 18 days at ≤-15°C was confirmed during the range finding study for the two-generation study (Project 503366).

Storage conditions: Diets were kept at room temperature in the diet store room in the animal house.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed on one day during the treatment period according to a validated method (Project 503368). Samples of diets were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations).
Random samples from all diet preparations were taken in the two-generation study (Project 503367) and stored at ≤-15°C for possible future analysis. No additional reserve samples were taken separately for the present study. Any remaining samples were discarded at finalization of the study report.
The accuracy of diet preparations was considered acceptable if the mean measured concentrations were 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.

Details on mating procedure:

One female was placed on a one-to-one-basis in the cage of a male rat. Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the female was separated from the male. When sufficient mated females had been obtained for each dose group, the surplus females were removed from the study.

Duration of treatment / exposure:

Days 6 to 20 post-coitum, inclusive.

Frequency of treatment:

Ad libitum from Days 6 to 20 post-coitum, inclusive.

Duration of test:

Days 6 to 20 post-coitum, inclusive.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 1500, 5000, 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

22 females per dose group

Control animals:

yes, plain diet

Details on study design:

Rationale: This species and strain of rat has been recognized as appropriate for developmental toxicity studies. WIL Research Europe B.V. has historical data on the background incidence of fetal malformations and developmental variations in this species from the same strain and source.
This animal model has been proven to be susceptible to the effects of developmental toxicants.

Rationale for dose levels: Dose levels were selected based on results of the dose range finding study.

Examinations

Maternal examinations:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from Day 0 post-coitum onwards up to the day prior to necropsy. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored. In the data tables, the scored grades were reported, as well as the percentage of animals affected in summary tables.

Body weights: Days 0, 3, 6, 9, 12, 15, 17 and 20 post-coitum.

Food consumption: Days 0-3, 3-6, 6-9, 9-12, 12-15, 15-17 and 17-20 post-coitum.

Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

Necropsy: All animals were sacrificed on Day 20 post-coitum using an oxygen/carbon dioxide procedure (approximately 40/60% oxygen/carbon dioxide respectively) and subsequently subjected to a limited post mortem examination, including an macroscopic examination of the cranial, thoracic and abdominal tissues and organs, with special attention being paid to the reproductive organs. All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).
All macroscopic abnormalities were recorded, collected and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands).

Organ weights: The following organ weights and terminal body weight were recorded from all pregnant females on the scheduled day of necropsy:
Liver
Spleen
After weighing, samples of these organs were fixed in 10% buffered formalin for possible future analysis.

Ovaries and uterine content:

Each ovary and uterine horn of animals surviving to planned necropsy was dissected and examined as quickly as possible to determine:
- The number of corpora lutea.
- The weight of the (gravid) uterus.
- The number and distribution of live and dead fetuses.
- The number and distribution of embryo-fetal deaths.
- The weight of each fetus.
- The sex of each fetus from the ano-genital distance (during necropsy) and also from gonadal inspections (during further fetal examination).
- Externally visible macroscopic fetal abnormalities.
In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites (Salewski staining prepared at WIL Research Europe using Ammoniumsulfide-solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)).
The female genital tract including the placentas was preserved in 10% buffered formalin for possible histopathological examination.

Fetal examinations:

External, visceral and skeletal fetal findings were recorded as developmental variations or malformations.

External: Each viable fetus was examined in detail, weighed and sexed. All live fetuses were euthanized by administration of approximately 0.05 mL (=10mg) of sodium pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) into the oral cavity using a small flexible plastic or metal feeding tube. The crown-rump length of late resorptions was measured, the degree of autolysis recorded, a gross external examination performed (if possible). Late resorptions with malformations were fixed in 10% buffered formalin.

Visceral (Internal): All fetuses were examined for visceral anomalies by dissection in the fresh (non-fixed) state. The thoracic and abdominal cavities were opened and dissected using a technique described by
Stuckhardt and Poppe. This examination included the heart and major vessels. Fetal kidneys were examined and graded for renal papillae development as described by Woo and Hoar. The sex of all fetuses was confirmed by internal examination.
The heads were removed from approximately one-half of the fetuses in each litter and placed in Bouin's solution (Klinipath, Duiven, The Netherlands). Tissues were then transferred to a 70% aqueous ethanol solution for subsequent processing and soft-tissue examination using the Wilson sectioning technique. After examination, the tissues were stored in 10% buffered formalin.
All carcasses, including the carcasses without heads, were eviscerated and fixed in identified containers containing 96% aqueous ethanol (Klinipath, Duiven, The Netherlands) for subsequent examination of skeletons.

Skeletal: Each eviscerated fetus, following fixation in 96% aqueous ethanol, was macerated in potassium hydroxide (Merck, Darmstadt, Germany) and stained with Alizarin Red S (Klinipath, Duiven, The Netherlands) by a method similar to that described by Dawson (Ref. 5). The skeletal examination was done following this procedure on the fetuses from all groups.
The specimens were archived in glycerin (Klinipath, Duiven, The Netherlands) with bronopol (Alfa Aesar, Karlsruhe, Germany) as preservative.
A few bones were not available for skeletal examination because they were accidentally damaged or lost during processing. The missing bones were listed in the raw data; evaluation by the fetal pathologist and study director determined there was no influence on the outcome of the individual or overall skeletal examinations, or on the integrity of the study as a whole.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control group.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Mann Whitney test was used to compare mean litter proportions (percent of litter) of the number of viable and dead fetuses, early and late resorptions, total resorptions, pre- and postimplantation loss, and sex distribution.
- Mean litter proportions (percent per litter) of total fetal malformations and developmental variations (external, visceral and skeletal), and each particular external, visceral and skeletal malformation or variation were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test was used to compare the compound-treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations might be rounded off before printing. Therefore, two groups might display the same printed means for a given parameter, yet display different test statistics values.

No statistics were applied for data on maternal survival, pregnancy status, group mean numbers of dead fetuses, early and late resorptions, and pre- and post-implantation loss.

Indices:

For each litter the following calculations were performed:

(number of corpora lutea – number of implantation sites)
Pre-implantation loss (%) = ------------------------------------------------------------ x 100
Number of corpora lutea

(number of implantation sites – number of live foetuses)
Post-implanatation loss (%) = ----------------------------------------------------------- x 100
Number of implantation sites

The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of foetuses and the number of litters available for examination in the group; and 2) considering the litter as the basic unit for comparison, calculating the number of affected foetuses as a men litter proportion on a total group basis, where:

Number of viable foetuses affected/litter
Viable foetuses affected/liiter (%) = --------------------------------------------- x 100
Number of viable foetuses/litter

Historical control data:

Historical data used for Fetal Morphology.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
Mortality: There were no unscheduled deaths during the study.

Clinical signs: No clinical signs were noted for any animal.

Body weights: At 15000 ppm, animals had reduced body weight gain from Day 9 post coitum onwards. Body weight gain corrected for the gravid uterus weight was also lower for these animals.
Females at this dose level also had slightly heavier absolute body weights on Day 0. As treatment commenced on Day 6 post coitum, the difference from controls was attributable to chance.

Food consumption: There were no toxicologically relevant effects on food consumption with treatment. At 15000 ppm, relative food consumption was lower from Days 9-12 of the post coitum period. As this was transient, it was not considered to be adverse.
Food consumption was higher than controls for females at 5000 and 15000 ppm on post coitum Days 12-15 (absolute and relative) and 17-20 (5000 ppm = absolute only, 15000 ppm = relative only). This was not considered to be toxicologically relevant, however, as values remained within normal limits, and a reduction in food consumption would be more likely if a toxicologically relevant effect on food consumption were present.

Test article intake: Dose levels of 1500, 5000 and 15000 ppm corresponded to a mean test article intake of 128, 439 and 1266 mg/kg/day, respectively.

Macroscopic examination: There were no toxicologically relevant effects on macroscopic findings noted with treatment up to 15000 ppm.
At 5000 ppm, an enlarged liver and uterus containing fluid were noted for individual animals. These were incidental and had no relationship with treatment.

Organ weights: Liver and spleen weights were similar between controls and treated animals.

Maternal pregnancy data: There were 21, 18, 18 and 21 pregnant females at 0, 1500, 5000 and 15000 ppm, respectively.
There were no differences between treated animals and controls in the number of corpora lutea, implantation sites, viable or dead fetuses, early or late resorptions, or in pre- or post-implantation loss.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

15 000 ppm (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter size: There were no effects of treatment up to 15000 ppm on litter size.
Mean litter sizes were 11.6, 10.8, 12.3 and 11.1 fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively.

Sex ratio: The sex ratio was unaffected with treatment up to 15000 ppm.

Fetal body weight: There were no treatment related effects on fetal body weight up to 15000 ppm.
Mean (combined) fetal body weights for the control, 1500, 5000 and 15000 ppm were 3.6, 3.7, 3.7, 3.6 grams, respectively.

Fetal morphological examinations: The numbers of fetuses (litters) available for external, visceral and skeletal morphological evaluation were 243 (21), 195 (18), 222 (18), 233 (21) in the control, 1500, 5000 and 15000 ppm groups, respectively. Soft cephalic tissue examinations were done for approximately half of the fetuses in each group. Malformations were observed in 1 (1) 1 (1), 1 (1) and 5 (4) fetuses (litters) in the control, 1500, 5000 and 15000 ppm groups, respectively.

External malformations and variations: There were no treatment related effects on fetal external morphology up to 15000 ppm.
A small lower jaw, an external malformation, was observed for a single control fetus. As this was seen for a control animal, this was not attributable to treatment. There were no other malformations and no external variations were seen for any fetuses.

Visceral malformations and variations: There were no treatment related effects on fetal visceral morphology.
One fetus at 15000 ppm had an absent eye. At the single incidence observed, this was not considered to be treatment related.
Visceral variations were observed for 7.7, 9.1, 6.8 and 8.2% of the fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively. Visceral variations included small supernumerary lobe of the liver, discolored adrenal gland, dilated and/or convoluted ureters, liver appendix, and partially undescended thymus horn(s). These were not considered attributable to treatment as they occurred at similar frequencies across the groups, occurred infrequently, did not follow a dose-related trend and/or occurred at frequencies within the range of available historical control data.

Skeletal malformations and variations: There were no toxicologically relevant effects on fetal skeletal morphology.
Skeletal malformations, consisting of bent limb bones (abnormal curvature of the limb bones), were seen for 0(0), 0(0), 0(0) and 4(3) fetuses (litters) in the control, 1500, 5000 and 15000 ppm groups, respectively. This corresponds to an incidence of 1.6% in the 15000 ppm group. One of these fetuses, A088-02, had multiple bones with abnormalities (including the clavicle, femur, fibula, humerus, ilium, ischium, radius, scapula, tibia and ulna). However, these abnormalities were at least partially attributable to his low body weight (2.8 gram) and the resulting developmental delay there from. This fetus was the exception as the other affected fetuses had bent limb bones representative of those previously seen in the historical control data. Though the incidence was significantly higher than controls, it remained below the maximum of 2% seen in the historical control database. Taken together, the bent limb bones were not considered to be treatment related.
Two other skeletal malformations were noted. Sternoschisis was seen for one fetus at 1500 ppm (incidence of 0.4%) and a malpositioned metacarpal was noted for a single fetus at 5000 ppm (incidence of 0.7%). As these malformations occurred for individual fetuses and remained under the maximum incidence noted in the historical control database, they were not considered to be treatment related.
Skeletal variations were observed in 76.3, 90.0, 83.1 and 84.4% of fetuses in the control, 1500, 5000 and 15000 ppm groups, respectively. An increased incidence of bent ribs was seen for fetuses of all treatment groups. Bent ribs were seen for 21.5, 19.3 and 21.1% of fetuses at 1500, 5000 and 15000 ppm, respectively, while this was seen for 6.2% of control fetuses. The increase was not considered to be toxicologically relevant as it occurred in the absence of a dose-dependent distribution and remained within the normal range of historical control data. Furthermore, the toxicological relevance of bent ribs is debatable since these are almost completely reversible during the pre-weaning period. As such, the higher incidence of bent ribs for treated animals was not considered to be toxicologically relevant.
Other skeletal variations seen in the control and/or treated groups included 14th rudimentary ribs, ossification of cervical centrum #1, 14th full ribs, reduced ossification of the vertebral centra, caudal shift of the pelvic girdle, reduced ossification of the skull, unossified/reduced ossification of the pubis, and unossified sternebrae nos. 1,2,3 and/or 4, malaligned sternebrae, branched sternebrae, generally reduced ossification of the entire skeleton, partially fused zygomatic arch, reduced ossification of the vertebral arches, unossification of the hyoid body, 7th cervical rudimentary or full ribs, ossified tarsals, . These variations were not considered to be treatment related because they occurred at similar frequencies in the control and treatment groups and/or remained within the historical control range.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

15 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no toxicologically relevant effects on developmental parameters with treatment upto 15000 ppm.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

BODY WEIGHTS (GRAM) SUMMARY FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
POST COITUM
DAY 0	MEAN ST.DEV. N	204 7.6 21	210 8.1 18	207 8.2 18	212** 10.0 21
DAY 3	MEAN ST.DEV. N	219 8.5 21	225 10.4 18	222 10.7 18	226 10.8 21
DAY 6	MEAN ST.DEV. N	225 8.6 21	232 11.2 18	230 9.1 18	232 12.5 21
DAY 9	MEAN ST.DEV. N	237 10.2 21	244 11.7 18	241 12.1 18	238 16.4 21
DAY 12	MEAN ST.DEV. N	251 11.3 21	256 11.9 18	256 11.3 18	249 16.4 21
DAY 15	MEAN ST.DEV. N	265 12.5 21	269 13.0 18	268 12.5 18	261 17.1 21
DAY 17	MEAN ST.DEV. N	286 14.8 21	290 15.6 18	289 13.9 18	281 18.3 21
DAY 20	MEAN ST.DEV. N	325 20.0 21	328 19.1 18	332 18.0 18	318 25.8 21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

BODY WEIGHT GAIN (%) SUMMARY FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
POST COITUM
DAY 0	MEAN ST.DEV. N	-10 2.2 21	-9 2.0 18	-10 2.1 18	-9 1.9 21
DAY 3	MEAN ST.DEV. N	-3 1.9 21	-3 1.7 18	-3 2.2 18	-2 1.9 21
DAY 6	MEAN ST.DEV. N	0 0.0 21	0 0.0 18	0 0.0 18	0 0.0 21
DAY 9	MEAN ST.DEV. N	5 2.2 21	5 2.0 18	5 2.2 18	3** 2.8 21
DAY 12	MEAN ST.DEV. N	12 2.4 21	11 1.9 18	11 1.8 18	7** 2.6 21
DAY 15	MEAN ST.DEV. N	18 3.0 21	16 2.1 18	17 3.0 18	12** 3.1 21
DAY 17	MEAN ST.DEV. N	27 4.0 21	25 2.8 18	26 2.8 18	21** 4.4 21
DAY 20	MEAN ST.DEV. N	44 6.1 21	42 4.9 18	45 4.6 18	37** 6.9 21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

FOOD CONSUMPTION (G/ANIMAL/DAY) SUMMARY FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
POST COITUM
DAYS 0 – 3	MEAN ST.DEV. N	20 1.5 21	20 1.7 18	20 1.7 18	20 2.3 21
DAYS 3 – 6	MEAN ST.DEV. N	21 2.0 21	22 2.3 18	22 2.1 18	22 2.4 21
DAYS 6 – 9	MEAN ST.DEV. N	21 3.1 21	22 2.1 18	22 2.1 18	20 3.3 21
DAYS 9 – 12	MEAN ST.DEV. N	22 2.7 21	23 2.6 18	23 2.5 18	20 4.5 21
DAYS 12 – 15	MEAN ST.DEV. N	23 2.4 21	24 1.8 18	25* 2.3 18	25* 2.8 21
DAYS 15 – 17	MEAN ST.DEV. N	24 3.1 21	24 2.6 18	24 2.8 18	24 2.4 21
DAYS 17 – 20	MEAN ST.DEV. N	24 2.2 21	25 2.5 18	27* 2.5 18	25 3.5 21
MEAN OF MEANS		22	23	23	22

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

TEST ARTICLE INTAKE (MG SUBSTANCE/KG BODY WEIGHT/DAY) SUMMARY FEMALES

F0-GENERATION

		GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
POST COITUM
DAYS 6 – 9	MEAN ST.DEV. N	0 0.0 21	133 10.6 18	460 38.3 18	1245 184.0 21
DAYS 9 -12	MEAN ST.DEV. N	0 0.0 21	132 11.1 18	451 39.3 18	1172 228.1 21
DAYS 12 – 15	MEAN ST.DEV. N	0 0.0 21	133 9.0 18	463 29.7 18	1424 149.8 21
DAYS 15 – 17	MEAN ST.DEV. N	0 0.0 21	125 9.9 18	422 40.9 18	1289 100.8 21
DAYS 17 -20	MEAN ST.DEV. N	0 0.0 21	115 8.6 18	299 27.1 18	1201 128.6 21
MEAN OF MEANS		0	128	439	1266

Explanation for excluded data are listed in the tables of the individual values (in the full study report).

 

MACROSCOPIC FINDINGS SUMMARY FEMALES

	GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
END OF TREATMENT
Animal examined	22	22	22	22
Animals without findings	22	22	20	22
Animals affected	0	0	2	0
Liver Enlarged	0	0	1	0
Uterus Contains fluid	0	0	1	0

#/## Fisher’s Exact test significant at 5% (#) or 1% (##) level

 

ORGAN WEIGHTS (GRAM) SUMMARY FEMALES

		GROUP 1 CONTROL	GROUP 2 1500 PPM	GROUP 3 5000 PPM	GROUP 5 15000 PPM
END OF TREATMENT
BODY W. (GRAM)	MEAN ST.DEV. N	321 19 21	323 18 18	327 18 18	314 25 21
LIVER (GRAM)	MEAN ST.DEV. N	14.71 1.76 21	14.86 1.64 18	14.96 1.34 18	14.59 1.52 21
SPLEEN (GRAM)	MEAN ST.DEV. N	0.580 0.103 21	0.597 0.092 18	0.568 0.066 18	0.573 0.066 21

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Conclusions:

In conclusion, based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (Weston 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.

Executive summary:

Title

A prenatal developmental toxicity study of DVS 005 (WESTON 705T) in rats by dietary administration.

 

Guidelines

The study procedures described in this report were based on the following guidelines:

1) Organization of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 414, Prenatal Developmental Toxicity Study, January 2001.

2) Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.31: "Prenatal Developmental Toxicity Study". Official Journal of the European Union No. L142, May 2008.

3) The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998.

 

Rationale for dose levels

Dose levels were selected based on results of the dose range finding study.

 

Study outline

Eighty-eight mated female Wistar Han rats were assigned to four dose groups. The test item was administered by inclusion in the diet from Days 6 to 20 post-coitum at doses of 1500, 5000, 15000 ppm (Groups 2, 3 and 4 respectively) corresponding to a mean test article intake of 128, 439 and 1266 mg/kg/day, respectively. The rats of the control group received the same diet without the test substance. Females were checked daily for the presence of clinical signs. Food consumption and body weight were determined at periodic intervals. Prepared diet was analyzed once for accuracy and homogeneity.

 

All animals surviving to Day 20 post-coitum were subjected to an examination post-mortem and external, thoracic and abdominal macroscopic findings were recorded. A laparohysterectomy was performed on each surviving female of the groups. The uteri, placentae and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations and corpora lutea were recorded. Gravid uterine weights were recorded, and corrected body weights (changes) were calculated. Liver and spleen weights were also recorded. The fetuses were weighed, sexed and examined for external, visceral and skeletal malformations and developmental variations. All live fetuses were euthanized. One half of the fetuses were decapitated and the heads were fixed in Bouin’s fixative, all fetuses were dissected and examined for visceral anomalies and subsequently fixed in 96% aqueous ethanol and stained with Alizarin Red S for skeletal examinations.

 

RESULTS

Accuracy and homogeneity of diet preparations were demonstrated by analyses.

 

Maternal findings

Females at 15000 ppm had lower body weight gains from Day 9 post coitum onwards.

There were no other effects on maternal parameters up to 15000 ppm and no maternal toxicity was observed in the 1500 or 5000 ppm groups.

 

Developmental findings

There were no toxicologically relevant effects on developmental parameters with treatment up to 15000 ppm.

 

CONCLUSION

Based on the results in this prenatal developmental toxicity study the maternal No Observed Adverse Effect Level (NOAEL) for DVS 005 (WESTON 705T) was established as 5000 ppm and the developmental NOAEL was established as at least 15000 ppm. Dose levels of 5000 and 15000 ppm correspond to a mean test article intake of 439 and 1266 mg/kg/day, respectively.