Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(Jan. 2001)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Version / remarks:

(May 2008)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

(August 1998)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

EC Number:

278-355-8

EC Name:

Diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide

Cas Number:

75980-60-8

Molecular formula:

C22H21O2P

IUPAC Name:

(mesitylcarbonyl)(diphenyl)phosphine oxide

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approximately 10 to 14 weeks
- Weight at study initiation:
- Housing: individually in Macrolon plastic cages (MIII type)
- Diet (e.g. ad libitum): ad lib. (SM R/M-Z from SSNIFF)
- Water (e.g. ad libitum): ad lib. (tap water)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24°C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10 aph
- Photoperiod (hrs dark / hrs light): 12h / 12h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

1% in water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 5 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test item, vehicle, and/or formulation. No correction was made for the purity of the test item.


VEHICLE
- Justification for use and choice of vehicle (if other than water): solubility
- Concentration in vehicle: 10, 30, 100 mg/ml
- Amount of vehicle (if gavage): 5ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dose preparations were taken at the test facility on a single occasion during the treatment period (formulations were prepared and sampled on 12 October 2015). The samples were dispatched on dry ice to ABL where they were analyzed to assess accuracy of preparation (all groups), homogeneity (lowest and highest concentration) and stability in vehicle over 5 hours at room temperature (lowest and highest concentration).

- No test item was detected in the Group 1 formulations.
- The concentrations analyzed in the formulations of Group 2, 3 and 4 were in agreement with the target concentrations (i.e. mean accuracies between 85% and 115%).
- The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
- Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 5 hours (i.e. relative difference ≤ 10%).

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From Days 6 to 20 post-coitum, inclusive

Frequency of treatment:

Once daily for 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

22 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected by the Sponsor and based on data from a maternal toxicity study, (BASF Project 10R0490/03X053, WIL Project 510610). In this study, the pregnant female rats treated at 500 mg/kg bw/day showed reduced body weight gain arising from reduced food consumption. 5/6 animals showed hunched posture, piloerection and/or salivation and increased liver weights were apparent at termination. No clear signs of toxicity were observed in pregnant female rats treated at 150 mg/kg bw/day.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Days 2, 6, 9, 12, 15, 18 and 21 post-coitum.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated
- Time schedule for examinations: Days 2-6, 6-9, 9-12, 12-15, 15-18 and 18-21 post-coitum

WATER CONSUMPTION: Subjective appraisal was maintained during the study, but no quantitative assessment was introduced.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21 post-coitum (or within 24h of abortion or early delivery)
- Organs examined: All macroscopic abnormalities (external, thoracic, abdominal) were recorded, collected and fixed in 10% buffered formalin

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 and distribution of implantations: Yes (In case implantations were not macroscopically visible, the uterus was stained using the Salewski technique in order to determine any former implantation sites )
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: sex and weight of each fetus, weight of each placenta (of live fetuses only)

Fetal examinations:

- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: Yes: [half per litter, i.e., the fetuses also used for visceral examination]

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 (Ref. 6) (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 7(many-to-one rank test)) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 8) was applied to frequency data.
- The Mann Whitney test (Ref. 9) 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 post-implantation 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 (Ref. 10) to determine intergroup differences. If the ANOVA revealed statistically significant (p<0.05) intergroup variance, Dunn’s test (Ref. 11) 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:

Pre-implantation loss = (number of corpora lutea - number of implantation sites) / number of corpora lutea
Post-implantation loss = (number of implantation sites - number of live fetuses) / number of implantation sites
Viable fetuses affected/litter = number of viable fetuses affected/litter / number of viable fetuses/litter

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Five females delivered early on Day 21 post-coitum (one vehicle control, one 50 mg/kg bw/day treated female, two 150 mg/kg bw/day treated females and one 500 mg/kg bw/day treated female). Additionally, one female in the 500 mg/kg bw/day group had an early delivery on Day 20 post-coitum.

The incidence and persistence of salivation showed a dose related increase. In 14/22 females treated with 150 mg/kg bw/day salivation was observed for one to a few days at the end of treatment. For one to several days, salivation was noted in 19/22 females in the highest dose group (500 mg/kg bw/day). Since no correlated findings were noted, this was attributed to the taste of the test item and of no toxicological relevance.

Furthermore, piloerection in 7/22 females and hunched posture in 4/22 females was observed for one to several days in females treated at 500 mg/kg bw.day.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain was significantly reduced in the highest dose group compared to vehicle controls from Day 9 post-coitum onwards. Consequently, on Day 21 post-coitum mean body weights and weight gain corrected for uterus weight were significantly lower in 500 mg/kg bw/day treated females compared to the controls (mean body weight on day 21 post coitum was 285 g compared to 305 g in controls). Body weights and body weight gain were unaffected at 50 and 150 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At the beginning of treatment (Day 6 to Day 12 post-coitum), food consumption was significantly reduced in Group 4 compared the control Group (maximum approximately 20%). This fully recovered to similar levels as controls from Day 12 post-coitum onwards. Food consumption was unaffected at 50 and 150 mg/kg bw/day.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted that were considered to be treatment related.

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.

Early or late resorptions:

no effects observed

Description (incidence and severity):

There were no effects on the number of pregnant females, corpora lutea, implantation site and pre- or post-implantation loss noted with treatment up to 500 mg/kg bw/ day.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Four animals were found not pregnant, (two 50 mg/kg bw/day treated females, one 150 mg/kg bw/day treated female and one 500 mg/kg bw/day treated female). All other females were pregnant and had litters with viable fetuses.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, female fetal weights were slightly, but significantly lower compared to the control group (4.8 gram versus 5.1 gram in controls). A similar, but not significant, reduction was observed in male fetal weights (5.0 gram versus 5.3 gram in controls) and consequently combined fetal weights.
The placenta weights were unaffected by treatment up to 500 mg/kg bw/day.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The male:female ratio was unaffected by treatment up to 500 mg/kg bw/day.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter size was unaffected by treatment up to 500 mg/kg bw/day. The numbers of fetuses (litters) available for morphological examination on Day 21 post-coitum were 233 (22), 200 (20), 202 (21) and 202 (20) in Groups 1, 2, 3, and 4, respectively.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related effects on external morphology following treatment up to 500 mg/kg bw/day. The only external malformations observed, were noted in two fetuses from a litter at 500 mg/kg bw/day. Fetus A086-05 had no tail and fetus A086-06 had a tail that was filamentous, which was confirmed at skeletal examination. Both these tail malformations were not seen previously among historical controls, however, because they pertain to the same group of tail abnormalities and occurred in one litter, they can be considered genetic in origin and are not related to treatment.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was an increase in the number of fetuses with bent limb bones in the highest dose group. Ten fetuses from 5 litters were affected with this skeletal malformation, compared to one control fetus (bent limb bones were not observed at 50 and 150 mg/kg bw/day). In all cases one or both scapulae were bent and in three fetuses also humeri were involved. The incidence of bent limb bones at 500 mg/kg bw/day was far above the upper limit in historical control fetuses (10.6% versus 0.7% per litter, respectively) and therefore the bent limb bones were considered to be treatment related. The higher incidence of bent limb bones at 500 mg/kg bw/day coincided with an increased litter incidence for bent ribs in this group. Mean litter proportions for this skeletal variation were 13.5%, 23.5%, 22.1% and 69.9% per litter in Group 1, 2, 3 and 4, respectively. The incidence in Group 4 was statistically significantly increased and all fetuses with bent limb bones also had bent ribs.

At 500 mg/kg bw/day, the incidence of the skeletal variations reduced ossification of the skull and unossified metatarsals and metacarpals were significantly increased compared to the control group. Mean litter incidences for reduced ossification of skull bones were 12.4%, 12.5%, 21.1%, 45.9% and for unossified metatarsals and metacarpals 5.4%, 6.6%, 3.2% and 21.0% per litter in the control, 50, 150 and 500 mg/kg bw/day group, respectively. Both these parameters are indicative of retarded skeletal ossification which is in line with the lower fetal body weights in this dose group. Both findings are considered to be related to the decreased maternal body weights (-7%).

Visceral malformations:

no effects observed

Description (incidence and severity):

There were no treatment related effects on visceral morphology following treatment up to 500 mg/kg bw/day.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Fetal abnormalities

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: forelimb

skeletal: hindlimb

Description (incidence and severity):

See above

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

In the abscence of gross limb malformations, and in the presence of retardation as a consequence of maternal toxicity, bent limb bones could be considered temporary variations rather than malformations. It is hypothesized, that during development the increase in muscle mass puts stress on the bones. If ossification is delayed, the bones might not be able to counteract this pressure and appear bowed until ossification is finalized. Bone development in rats continues long after birth, extending into young adulthood. In a few studies, pups were followed sequentially after birth, and bent long bones and scapulae were transient in nature and appeared normal by the time of weaning.

References:

- De Schaepdrijver L., Delille P., Geys H., Boehringer-Shahidi C., Vanhove C. In vivo longitudinal micro-CT study of bent long limb bones in rat offspring. Reprod Toxicol 46, 91–97 (2014).

- Mitchard T.L., French J. Apparent postnatal recovery of chondrodystrophy in the Harlan Han Wistar rat. Reprod Toxicol 32, 169–70 (2011).

- Kimmel C.A., Garry M.R., DeSesso J.M. Review Articel: Relationship Between Bent Long Bones, Bent Scapulae, and Wavy Ribs: Malformations or Variations? Birth Defects Research (Part B) 101:379–392 (2014)